Anti-factor XI monoclonal antibodies and methods of use thereof

ABSTRACT

Compositions and methods for inhibiting thrombosis without compromising hemostasis are described. Compositions include anti-factor XI monoclonal antibodies (aXIMabs) capable of binding to an epitope on the heavy chain of human FXI, particularly the A3 domain of the heavy chain of human FXI. Compositions also include epitope-binding fragments, variants, and derivatives of the monoclonal antibodies, cell lines producing these antibody compositions, and isolated nucleic acid molecules encoding the amino acid sequences of the antibodies. The disclosure further includes pharmaceutical compositions comprising the disclosed anti-factor XI monoclonal antibodies, or epitope-binding fragments, variants, or derivatives thereof, in a pharmaceutically acceptable carrier. Methods of the disclosure include administering the compositions described above to a subject in need thereof for the purpose of inhibiting thrombosis, reducing a required dose of an antithrombotic agent in the treatment of thrombosis, treating metastatic cancer, or treating an acute inflammatory reaction.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation of U.S. patent application Ser. No. 15/470,758,filed Mar. 27, 2017, which is a continuation of U.S. application Ser.No. 14/809,551, filed Jul. 27, 2015, now U.S. Pat. No. 9,636,399, issuedMay 2, 2017, which is a continuation of U.S. application Ser. No.14/242,692, filed Apr. 1, 2014, now U.S. Pat. No. 9,125,895, issued Sep.8, 2015, which is a continuation of U.S. application Ser. No.13/783,952, filed Mar. 4, 2013, now abandoned, which is a divisional ofU.S. application Ser. No. 13/446,320, filed Apr. 13, 2012, now U.S. Pat.No. 8,399,648, issued Mar. 19, 2013, which is a divisional of U.S.application Ser. No. 12/744,037, filed Oct. 13, 2010, now U.S. Pat. No.8,236,316, issued Aug. 7, 2012, which is the U.S. National Stage ofInternational Application No. PCT/US2008/084336, filed Nov. 21, 2008,published in English under PCT Article 21(2), which claims the benefitof U.S. Provisional Application No. 60/989,523, filed Nov. 21, 2007. Allof the above-listed applications are herein incorporated by reference intheir entirety.

FIELD OF THE INVENTION

The present invention relates to antibodies capable of binding to factorXI and methods of use thereof, particularly methods of use asantithrombotic agents that do not compromise hemostasis.

BACKGROUND OF THE INVENTION

Hemostasis is a vital function that stops bleeding and protects theintegrity of blood circulation on both molecular and macroscopic levels.Hemostasis includes a coagulation cascade of sequentially activatableenzymes that is traditionally divided into three parts: 1) an intrinsicpathway, which includes interactions of blood coagulation proteins thatlead to the generation of coagulation factor IXa without involvement ofcoagulation factor VIIa; 2) an extrinsic pathway, which includesinteractions of blood coagulation proteins that lead to the generationof coagulation factor Xa and/or IXa without involvement ofthromboplastin antecedent (factor XI); and 3) a common coagulationpathway, including interactions of blood coagulation proteins II, V,VIII, IX and X that lead to the generation of thrombin (factor IIa).Thrombin activates platelets and generates fibrin, both of which areessential building elements of the hemostatic plug that is responsiblefor sealing the vascular breach. Complete absence of thrombin orplatelets causes paralysis of hemostasis and leads to lethal hemorrhage.

Thrombosis, like hemostasis, is a platelet and thrombin dependentprocess. Thrombosis is a pathological, intravascular,thrombin-dependent, progressive deposition of polymerized fibrin andactivated platelets that causes occlusion of blood vessels in variousorgans. In healthy mammals, intravascular coagulation is localized tothe site of hemostasis. Intraluminal progression into thrombosis isefficiently blocked by natural antithrombotic enzymes and inhibitors,such as activated protein C, plasmin, and antithrombin. Thrombosisdevelops when the antithrombotic system fails to control furtherintravascular thrombin generation. Causality of macrovascular and/ormicrovascular thrombosis in morbidity and mortality has been directlydocumented in various diseases that include deep vein thrombosis,pulmonary thromboembolism, peripheral artery thrombosis and embolism,retina vein thrombosis, as well as myocardial infarction (Meadows (1965)Med. J. Aust. 4:409-411; Harland (1966) Lancet 26:1158-1160), ischemicstroke (Carmon (1966) J. Neural. Sci. 4:111-119), anthrax sepsis andmeningococcal sepsis (Dalldorf (1977) Arch. Pathol. Lab. Med. 101:6-9),or heparin-induced thrombocytopenia (Rhodes (1973) Surg. Gynecol.Obstet. 136:409-416). Evidence for organ damage of thrombotic occlusionand hypoxic origin is also available in other disease groups, such ashemorrhagic fevers (Dennis (1969) Br. J. Haematol. 17:455-462; Gear(1979) Rev. Infect. Dis. 1:571-591; Ignatiev (2000) Immunol. Lett.71:131-140), diabetic angiopathy (Ishibashi (1981) Diabetes 30:601-606;Boeri (2001) Diabetes 50:1432-1439), kidney disease (Miller (1980)Kidney Int. 18:472-479; McCutcheon (1993) Lupus 2:99-103), and severalother conditions.

Although thrombosis and hemostasis are not identical molecularprocesses, they are similar enough that antithrombotic drugs developedto date inadvertently target both. Thrombosis is treated withantiplatelet, profibrinolytic, and anticoagulant agents, yet most ofthese agents can completely block both thrombosis and hemostasis whenadministered at their maximally effective doses. Antithrombotic drugseither target the building blocks of thrombi (fibrin and platelets) orinhibit molecules (coagulation factors) and cells (platelets) fromparticipating in the thrombus-forming process. It is widely believedamong clinicians and researchers that if an antithrombotic agent isunable to block hemostasis it will not work in thrombosis.

One of the oldest anticoagulant antithrombotic agents, heparin, is stillthe most widely given injection in the world. Sufficiently high doses ofheparin can achieve nearly 100% efficacy but only at the cost ofparalyzing hemostasis at such doses. Unfortunately, newer antithromboticagents, such as fractionated heparins or direct thrombin inhibitorsagents do not fare much better. As a result, antithrombotic agents,especially anticoagulants and profibrinolytic agents, must beadministered at less than their maximally efficacious doses, andthrombosis remains an under-treated disease. Introduction of newcompounds that are based on the promise of improved efficacy but areunable to promise improvement of hemostatic safety is unjustifiable. Todate, antithrombotic compounds have fallen short of promisingimprovement of safety. The ideal antithrombotic agent wouldanti-coagulate circulating blood without adversely affecting hemostasis.

Thus, there remains a pressing medical need for the development of safeyet efficacious agents that block intravascular thrombin generationwithout paralyzing hemostasis.

BRIEF SUMMARY OF THE INVENTION

Compositions and methods are provided for inhibiting thrombosis withoutcompromising hemostasis. Compositions include anti-factor XI monoclonalantibodies (aXIMabs) capable of binding to an epitope on the heavy chainof human FXI, particularly the A3 domain of the heavy chain of humanFXI. Compositions also include epitope-binding fragments, variants, andderivatives of the monoclonal antibodies, cell lines producing theseantibody compositions, and isolated nucleic acid molecules encoding theamino acid sequences of the antibodies. The invention further includespharmaceutical compositions comprising the anti-factor XI monoclonalantibodies of the invention, or epitope-binding fragments, variants, orderivatives thereof, in a pharmaceutically acceptable carrier. Methodsof the invention comprise administering the compositions described aboveto a subject in need thereof for the purpose of inhibiting thrombosis,reducing a required dose of an antithrombotic agent in the treatment ofthrombosis, treating metastatic cancer, or treating an acuteinflammatory reaction. Methods for making an anti-factor XI monoclonalantibody, or epitope-binding fragments, variants, or derivativesthereof, are also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C. Local sampling model and thrombogenic device used toinitiate thrombus formation. (FIG. 1A) Qualitative schematic of thelocal sampling method (not to scale). (FIG. 1B) Scanning electronmicroscope image of uncoated, and collagen coated (FIG. 1C)expanded-polytetrafluoroethylene (ePTFE) clinical vascular graftmaterial.

FIG. 2. Inhibition of the FXI procoagulant activity after administrationof an anti-FXI monoclonal antibody (aXIMab). A single i.v. bolus (2mg/kg) of aXIMab was given over 5 min to 4 separate baboons. Plasmasamples were collected into 1/10^(th) v/v 3.2% citrate anticoagulant andtested in a one-stage clotting assay to assess the inhibition of FXI.Each time point represents the mean of 2-4 separate animals, with eachexceeding 95% inhibition through day 8. All clotting times normalized byday 11.

FIGS. 3A-3B. FXI inhibition reduces platelet and fibrin deposition oncollagen coated vascular grafts. Effects of FXI inhibition on (FIG. 3A)platelet and (FIG. 3B) fibrin deposition on collagen coated (4 mm i.d.)vascular grafts. Thrombogenic grafts were placed in untreated (n=8) oraXIMab treated (n=6) animals (>95% FXI inhibition). Blood was allowed toflow through the devices at a clamped rate of 100 ml/min, producing anaverage wall shear rate of 265 s⁻¹. Significance levels are ***P=0.001.Values are means±SEM.

FIGS. 4A-4E. FXI inhibition by aXIMab reduces thrombin generation andplatelet activation, while not affecting fibrinolysis. (FIGS. 4A, 4C)Local and (FIGS. 4C, 4D) systemic thrombin/antithrombin (TAT) andβ-thromboglobulin (β-TG) levels during thrombus formation, respectively.Blood samples were also tested for the fibrinolysis product dimer (FIG.4E). Local values are those taken from the near wall, low flow boundarylayer 1 cm distal to the growing thrombus over the course of 10 minprior to its designated time, while systemic samples were taken from thearterio/venous shunt proximal to the thrombogenic device. Zero timepoints in all groups are from samples taken systemically immediatelybefore each study. FXI inhibition dramatically reduced local thrombinformation and platelet activation, which translated into significantsystemic reductions in both TAT and β-TG compared with collagen controlsby 60 min. Significant fibrinolysis was not detected in eithernon-treated or aXIMab treated animals.

FIG. 5. FXI inhibition limits thrombus stability and prevents vasculargraft occlusion under high arterial shear. Effects of FXI inhibition andASA on platelet deposition on collagen coated (2 mm i.d.) vasculargrafts are shown. Collagen-coated vascular graft segments were placed inpermanent arterio-venous shunts in untreated (n=9), ASA treated (n=6),and aXIMab treated (n=5) animals. Blood was allowed to flow through thegrafts at a rate of 100 ml/min, producing an average initial wall shearrate of 2120 s⁻¹. The flow was maintained by the pulsatile arterialpressure until the graft occluded (defined as ≤20 ml/min flow rate).Thrombi that formed in the grafts in the aXIMab treated animals wereunstable and did not occlude the grafts during the 60 min-long studies.Significance levels are *P=0.05, **P<0.01 compared with non-treatmentcontrols, using the log-rank test with the non-occluded devices beingcensored. Values are means±SEM.

FIG. 6. Template bleeding time is normal in FXI inhibited baboons.Bleeding time was monitored repeatedly, on the volar surface of thelower arm, before and during treatment with aXIMab or ASA using anFDA-approved method and device (Surgicutt). ASA (n=10) prolonged thebleeding time while inhibition of FXI by aXIMab (n=18) did notdemonstrably impair hemostasis compared with no treatment controls(n=14). The average of bleeding time changes are shown ±SEM.Significance levels are **P≤0.01.

FIGS. 7A-7B. (FIG. 7A) Binding of anti-factor XI monoclonal antibody 1A6to Factor XI using Western Blots. The bands in the left panel show thatantibody 1A6 recognizes Factor XI in human and non-human primate plasma.Similar bands were not observed for Factor XI depleted or deficientsamples (right panel). (FIG. 7B) aXIMab prevented visible fibrinformation and reduced platelet accumulation in FXII-inhibited ordeficient human blood under flow.

FIGS. 8A-8C. Inhibition of FXI procoagulant activity afteradministration of aXIMab. A single intravenous bolus of aXIMab (2 mg/kg)was given over 5 min to a single baboon. Plasma samples were collectedinto citrate anticoagulant and tested for (FIG. 8A) FXI procoagulantactivity, FXI antigen (FXI:Ag), and (FIG. 8B) inhibitor levels over 4weeks, with each time point being the mean of duplicate measurements.The FXI:Ag ELISA was able to detect both free and complexed FXI. Sincethe Bethesda assay detected only free FXI inhibitor (aXIMab), FXI:Ag andFXI activity at low inhibitor levels did not correlate until allcomplexes were cleared from circulation. (FIG. 8C) Western blot of 1 μlNHP and NBP samples size fractionated by non-reducing 7.5% SDS-PAGE.Detection was with a polyclonal antibody against human FXI. The fivelanes on the right represent samples prior to (0) or 1, 6, 8, and 27days after infusion of aXIMab. Abbreviations: XI—100 ng purified humanFXI; NHP—normal human plasma; NBP—normal baboon plasma; XI-DP—FXIdeficient human plasma.

FIG. 9 shows Western Blots of the binding of anti-factor XI monoclonalantibody 1A6 to chimeric proteins of FXI and Prekallikrein (PK)involving the substitution of FXI domains with PK domains. As shown inthe right panel, monoclonal antibody 1A6 bound all FXI/PK chimerasexcept the chimera in which the A3 domain of FXI was substituted with aPK domain.

FIG. 10 shows Western Blots of the binding of anti-factor XI monoclonalantibody 1A6 to chimeric proteins of FXI and PK involving thesubstitution of PK domains with FXI domains. Monoclonal antibody 1A6bound only to chimeras in which the A3 domain of FXI had been inserted.

FIG. 11 shows Western Blots of the binding of anti-factor XI monoclonalantibody 1A6 to recombinant FXI proteins involving mutations ofdifferent amino acids within the A3 domain. Although a polyclonalanti-factor XI antibody bound to all FXI A3 domain mutants, monoclonalantibody 1A6 did not bind to some mutants in which amino acids withinthe A3 domain were substituted with Alanine.

FIG. 12 shows the position of Alanine substitutions within the A3 domainof FXI that prevented or reduced the binding of monoclonal antibody 1A6to FXI.

FIGS. 13A-13D. (FIG. 13A) Aximab heavy chain nucleotide (SEQ ID NO: 2)and amino acid (SEQ ID NO: 3) sequences. (FIG. 13B) Aximab light chainnucleotide (SEQ ID NO: 4) and amino acid (SEQ ID NO: 5) sequences. (FIG.13C) CDR grafted Aximab heavy chain nucleotide (SEQ ID NO: 6) and aminoacid (SEQ ID NO: 7) sequences. (FIG. 13D) CDR grafted Aximab light chainnucleotide (SEQ ID NO: 8) and amino acid (SEQ ID NO: 9) sequences. CDRdefinitions and protein sequence numbering according to Kabat. CDRnucleotide and protein sequences are in bold. Changes from murinesequence are underlined in (FIG. 13C) and (FIG. 13D). Human acceptorgermline framework is IGHV2-5.

DETAILED DESCRIPTION I. Overview

The present invention relates to compositions and methods relating toanti-factor XI monoclonal antibodies (aXIMabs) for inhibiting thrombosiswithout compromising hemostasis. As described more fully below,monoclonal antibodies from a mouse cell line designated 1A6 wereprepared that recognized and bound to the heavy chain of primate(particularly human) factor XI (FXI), particularly to the Apple 3 (A3)domain of the heavy chain of human FXI. Although polyclonal anti-FXIantibodies are commercially available for research and diagnostic uses,these polyclonal antibodies are not safe to use in the clinic becausethey cannot be controlled and their production is dependent on theavailability of live animals (See, e.g., Gruber & Hanson (2003) Blood102:953-955). In contrast, monoclonal antibodies can be consistentlymanufactured in cell cultures, they can be synthetized, or purified frombyproducts of transgenic organisms that can range from fungi to plantsto animals. In addition, although mouse monoclonal anti-FXI antibodiesare currently available for research applications, no specificanti-factor XI monoclonal antibodies have been produced for use as safeantithrombotic agents in vivo.

One advantage of the anti-factor XI monoclonal antibodies of theinvention, or epitope-binding fragments, variants, or derivativesthereof, is their exceptional safety as anticoagulant agents. Theanti-factor XI monoclonal antibodies of the invention are monospecificfor FXI and do not inhibit essential proteins or interact with vitalmolecules and pathways, either in their intact form or when degraded.This is a particular safety advantage as compared to otherantithrombotic agents. Specifically, doses of anti-factor XI monoclonalantibody significantly exceeding the maximum effective dose (forexample, several times the maximum effective dose) did not produce anyadverse bleeding or other toxic side effects in subjects. In contrast,all other antithrombotic agents, such as vitamin K antagonists, indirectand direct inhibitors of essential coagulation enzymes, plateletinhibitors, fibrinolytic agents, and the like are ultimately dangerousand even fatal when they are administered at their maximally effectivedoses.

Other safety advantages of the anti-factor XI monoclonal antibodies ofthe invention as compared to other antithrombotic agents, such as smallmolecule enzyme inhibitors or platelet inhibitors, are that themonoclonal antibodies of the invention are metabolized and eliminatedwithout generation of toxic metabolic intermediates, and theirmetabolism is virtually independent of the integrity of liver and kidneyfunctions. In addition, the anti-factor XI monoclonal antibodies of theinvention do not interact with other pharmacological compounds and donot directly affect the activity or metabolism of other drugs, incontrast to other antithrombotic agents.

The anti-factor XI monoclonal antibodies of the invention are alsouseful as potent pharmacological agents. As described more fully below,anti-factor XI monoclonal antibody administration can prevent or treatdiseases where FXI activity contributes to pathology. The anti-factor XImonoclonal antibodies of the invention inhibit thrombus formationwithout causing bleeding and therefore provide a safe treatment optionfor thromboocclusive diseases in subjects, particularly humans. Anotheradvantage of anti-factor XI monoclonal antibody as compared to otherantithrombotic agents is that a single dose is safe and effective forlonger than one week. Most antithrombotic agents have shorter durationof action.

II. Definitions

It is to be noted that the term “a” or “an” entity refers to one or moreof that entity; for example, “an anti-factor XI monoclonal antibody” isunderstood to represent one or more anti-factor XI monoclonalantibodies. As such, the terms “a” (or “an”), “one or more,” and “atleast one” can be used interchangeably herein.

The term “hemostasis” as used herein refers to a coordinated mechanismthat maintains the integrity of blood circulation following injury tothe vascular system. In normal circulation without vascular injury,platelets are not activated and freely circulate. Vascular injuryexposes sub-endothelial tissue to which platelets can adhere. Adherentplatelets will attract other circulating platelets to form a preliminaryplug that is particularly useful in closing a leak in a capillary orother small vessel. These events are termed primary hemostasis. This is,typically, rapidly followed by secondary hemostasis that involves acascade of linked enzymatic reactions that result in plasma coagulationto reinforce the primary platelet plug. Accordingly, a hemostatic agentis any agent that slows or stops bleeding by promoting or enhancing anyof the physiological processes involved in hemostasis, includingcontraction of the blood vessels, adhesion and aggregation of formedblood elements, and blood or plasma coagulation.

The term “coagulation” as used herein refers to the process ofpolymerization of fibrin monomers, resulting in the transformation ofblood or plasma from a liquid to a gel phase. Coagulation of liquidblood may occur in vitro, intravascularly or at an exposed and injuredtissue surface. In vitro blood coagulation results in a gelled bloodthat maintains the cellular and other blood components in essentiallythe same relative proportions as found in non-coagulated blood, exceptfor a reduction in fibrinogen content and a corresponding increase infibrin. By “blood clot” is intended a viscous gel formed of, andcontaining all, components of blood in the same relative proportions asfound in liquid blood.

The term “coagulation cascade” as used herein refers to threeinterconnecting enzyme pathways as described, for example, by Manolin inWilson et al. (eds): Harrison's Principle of Internal Medicine, 14^(th)Ed. New York. McGraw-Mill, 1998, p. 341, incorporated herein byreference in its entirety. The intrinsic coagulation pathway leads tothe formation of Factor IXa, that in conjunction with Factors VIIIa andX, phospholipid and Ca²⁺ gives Factor Xa. The extrinsic pathway givesFactor Xa and IXa after the combination of tissue factor and factor VII.The common coagulation pathway interacts with the blood coagulationFactors V, VIII, IX and X to cleave prothrombin to thrombin (FactorIIa), which is then able to cleave fibrinogen to fibrin. As used herein,the term “polypeptide” is intended to encompass a singular “polypeptide”as well as plural “polypeptides,” and refers to a molecule composed ofmonomers (amino acids) linearly linked by amide bonds (also known aspeptide bonds). The term “polypeptide” refers to any chain or chains oftwo or more amino acids, and does not refer to a specific length of theproduct. Thus, peptides, dipeptides, tripeptides, oligopeptides,“protein,” “amino acid chain,” or any other term used to refer to achain or chains of two or more amino acids, are included within thedefinition of “polypeptide,” and the term “polypeptide” may be usedinstead of, or interchangeably with any of these terms. The term“polypeptide” is also intended to refer to the products ofpost-expression modifications of the polypeptide, including withoutlimitation glycosylation, acetylation, phosphorylation, amidation,derivatization by known protecting/blocking groups, proteolyticcleavage, or modification by non-naturally occurring amino acids. Apolypeptide may be derived from a natural biological source or producedby recombinant technology, but is not necessarily translated from adesignated nucleic acid sequence. It may be generated in any manner,including by chemical synthesis.

A polypeptide of the invention may be of a size of about 3 or more, 5 ormore, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 ormore, 200 or more, 500 or more, 1,000 or more, or 2,000 or more aminoacids. Polypeptides may have a defined three-dimensional structure,although they do not necessarily have such structure. Polypeptides witha defined three-dimensional structure are referred to as folded, andpolypeptides that do not possess a defined three-dimensional structure,but rather can adopt a large number of different conformations, arereferred to as unfolded. By an “isolated” polypeptide or a fragment,variant, or derivative thereof is intended a polypeptide that is not inits natural milieu. No particular level of purification is required. Forexample, an isolated polypeptide can be removed from its native ornatural environment. Recombinantly produced polypeptides and proteinsexpressed in host cells are considered isolated for purpose of theinvention, as are native or recombinant polypeptides that have beenseparated, fractionated, or partially or substantially purified by anysuitable technique.

Also included as polypeptides of the present invention are fragments,derivatives, analogs, or variants of the foregoing polypeptides, and anycombination thereof. The terms “fragment,” “variant,” “derivative,” and“analog” when referring to anti-factor XI monoclonal antibodies orantibody polypeptides of the present invention include any polypeptidesthat retain at least some of the antigen-binding properties of thecorresponding antibody or antibody polypeptide of the invention.Fragments of polypeptides of the present invention include proteolyticfragments, as well as deletion fragments, in addition to specificantibody fragments discussed elsewhere herein. Variants of anti-factorXI monoclonal antibodies and antibody polypeptides of the presentinvention include fragments as described above, and also polypeptideswith altered amino acid sequences due to amino acid substitutions,deletions, or insertions. Variants may occur naturally or benon-naturally occurring. Non-naturally occurring variants may beproduced using art-known mutagenesis techniques. Variant polypeptidesmay comprise conservative or non-conservative amino acid substitutions,deletions, or additions. Derivatives of anti-factor XI monoclonalantibodies and antibody polypeptides of the present invention, arepolypeptides that have been altered so as to exhibit additional featuresnot found on the reference antibody or antibody polypeptide of theinvention. Examples include fusion proteins. Variant polypeptides mayalso be referred to herein as “polypeptide analogs.” As used herein a“derivative” of an anti-factor XI monoclonal antibody or antibodypolypeptide refers to a subject polypeptide having one or more residueschemically derivatized by reaction of a functional side group. Alsoincluded as “derivatives” are those peptides that contain one or morenaturally occurring amino acid derivatives of the twenty standard aminoacids. For example, 4-hydroxyproline may be substituted for proline;5-hydroxylysine may be substituted for lysine; 3-methylhistidine may besubstituted for histidine; homoserine may be substituted for serine; andornithine may be substituted for lysine.

The term “polynucleotide” is intended to encompass a singular nucleicacid as well as plural nucleic acids, and refers to an isolated nucleicacid molecule or construct, e.g., messenger RNA (mRNA) or plasmid DNA(pDNA). A polynucleotide may comprise a conventional phosphodiester bondor a non-conventional bond (e.g., an amide bond, such as found inpeptide nucleic acids (PNA)). The term “nucleic acid” refers to any oneor more nucleic acid segments, e.g., DNA or RNA fragments, present in apolynucleotide. By “isolated” nucleic acid or polynucleotide is intendeda nucleic acid molecule, DNA or RNA, that has been removed from itsnative environment. For example, a recombinant polynucleotide encodingan anti-factor XI monoclonal antibody contained in a vector isconsidered isolated for the purposes of the present invention. Furtherexamples of an isolated polynucleotide include recombinantpolynucleotides maintained in heterologous host cells or purified(partially or substantially) polynucleotides in solution. Isolated RNAmolecules include in vivo or in vitro RNA transcripts of polynucleotidesof the present invention. Isolated polynucleotides or nucleic acidsaccording to the present invention further include such moleculesproduced synthetically. In addition, a polynucleotide or a nucleic acidmay be or may include a regulatory element such as a promoter, ribosomebinding site, or a transcription terminator.

As used herein, a “coding region” is a portion of nucleic acid thatconsists of codons translated into amino acids. Although a “stop codon”(TAG, TGA, or TAA) is not translated into an amino acid, it may beconsidered to be part of a coding region, but any flanking sequences,for example promoters, ribosome binding sites, transcriptionalterminators, introns, and the like, are not part of a coding region. Twoor more coding regions of the present invention can be present in asingle polynucleotide construct, e.g., on a single vector, or inseparate polynucleotide constructs, e.g., on separate (different)vectors. Furthermore, any vector may contain a single coding region, ormay comprise two or more coding regions, e.g., a single vector mayseparately encode an immunoglobulin heavy chain variable region and animmunoglobulin light chain variable region. In addition, a vector,polynucleotide, or nucleic acid of the invention may encode heterologouscoding regions, either fused or unfused to a nucleic acid encoding ananti-factor XI monoclonal antibody or fragment, variant, or derivativethereof. Heterologous coding regions include without limitationspecialized elements or motifs, such as a secretory signal peptide or aheterologous functional domain.

In certain embodiments, the polynucleotide or nucleic acid is DNA. Inthe case of DNA, a polynucleotide comprising a nucleic acid that encodesa polypeptide normally may include a promoter and/or other transcriptionor translation control elements operably associated with one or morecoding regions. An operable association is when a coding region for agene product, e.g., a polypeptide, is associated with one or moreregulatory sequences in such a way as to place expression of the geneproduct under the influence or control of the regulatory sequence(s).Two DNA fragments (such as a polypeptide coding region and a promoterassociated therewith) are “operably associated” if induction of promoterfunction results in the transcription of mRNA encoding the desired geneproduct and if the nature of the linkage between the two DNA fragmentsdoes not interfere with the ability of the expression regulatorysequences to direct the expression of the gene product or interfere withthe ability of the DNA template to be transcribed. Thus, a promoterregion would be operably associated with a nucleic acid encoding apolypeptide if the promoter was capable of effecting transcription ofthat nucleic acid. The promoter may be a cell-specific promoter thatdirects substantial transcription of the DNA only in predeterminedcells. Other transcription control elements, besides a promoter, forexample enhancers, operators, repressors, and transcription terminationsignals, can be operably associated with the polynucleotide to directcell-specific transcription. Suitable promoters and other transcriptionand translation control regions are well known in the art.

The present invention is directed to certain anti-factor XI monoclonalantibodies, or antigen-binding fragments, variants, or derivativesthereof. Unless specifically referring to full-sized antibodies such asnaturally occurring antibodies, the term “anti-factor XI monoclonalantibodies” encompasses full-sized antibodies as well as antigen-bindingfragments, variants, analogs, or derivatives of such antibodies, e.g.,naturally occurring antibody or immunoglobulin molecules or engineeredantibody molecules or fragments that bind antigen in a manner similar toantibody molecules.

The terms “antibody” and “immunoglobulin” are used interchangeablyherein. An antibody or immunoglobulin comprises at least the variabledomain of a heavy chain, and normally comprises at least the variabledomains of a heavy chain and a light chain. Basic immunoglobulinstructures in vertebrate systems are relatively well understood. See,e.g., Harlow et al. (1988) Antibodies: A Laboratory Manual (2nd ed.;Cold Spring Harbor Laboratory Press).

As will be discussed in more detail below, the term “immunoglobulin”comprises various broad classes of polypeptides that can bedistinguished biochemically. Those skilled in the art will appreciatethat heavy chains are classified as gamma, mu, alpha, delta, or epsilon,(γ, μ, α, δ, ε) with some subclasses among them (e.g., γ1-γ4). It is thenature of this chain that determines the “class” of the antibody as IgG,IgM IgA, IgD, or IgE, respectively. The immunoglobulin subclasses(isotypes) e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, etc., are wellcharacterized and are known to confer functional specialization.Modified versions of each of these classes and isotypes are readilydiscernable to the skilled artisan in view of the instant disclosureand, accordingly, are within the scope of the instant invention.Although all immunoglobulin classes are clearly within the scope of thepresent invention, the following discussion will generally be directedto the IgG class of immunoglobulin molecules. With regard to IgG, astandard immunoglobulin molecule comprises two identical light chainpolypeptides of molecular weight approximately 23,000 Daltons, and twoidentical heavy chain polypeptides of molecular weight 53,000-70,000Daltons. The four chains are typically joined by disulfide bonds in a“Y” configuration wherein the light chains bracket the heavy chainsstarting at the mouth of the “Y” and continuing through the variableregion.

Light chains are classified as either kappa or lambda (κ, λ). Each heavychain class may be bound with either a kappa or lambda light chain. Ingeneral, the light and heavy chains are covalently bonded to each other,and the “tail” portions of the two heavy chains are bonded to each otherby covalent disulfide linkages or non-covalent linkages when theimmunoglobulins are generated either by hybridomas, B cells, orgenetically engineered host cells. In the heavy chain, the amino acidsequences run from an N-terminus at the forked ends of the Yconfiguration to the C-terminus at the bottom of each chain.

Both the light and heavy chains are divided into regions of structuraland functional homology referred to as the “constant region” and the“variable region.” The terms “constant” and “variable” are usedfunctionally. In this regard, it will be appreciated that the variabledomains of both the light (V_(L)) and heavy (V_(H)) chain portionsdetermine antigen recognition and specificity. Conversely, the constantdomains of the light chain (C_(L)) and the heavy chain (C_(H)1, C_(H)2,or C_(H)3) confer important biological properties such as secretion,transplacental mobility, Fc receptor binding, complement binding, andthe like. By convention the numbering of the constant region domainsincreases as they become more distal from the antigen binding site oramino-terminus of the antibody. The N-terminal portion is a variableregion and at the C-terminal portion is a constant region; the C_(H)3and C_(L) domains actually comprise the carboxy-terminus of the heavyand light chain, respectively.

As indicated above, the variable region allows the antibody toselectively recognize and specifically bind epitopes on antigens. Thatis, the V_(L) domain and V_(H) domain, or subset of the complementaritydetermining regions (CDRs) within these variable domains, of an antibodycombine to form the variable region that defines a three dimensionalantigen binding site. This quaternary antibody structure forms theantigen binding site present at the end of each arm of the Y. Morespecifically, the antigen binding site is defined by three CDRs on eachof the V_(H) and V_(L) chains.

In naturally occurring antibodies, the six “complementarity determiningregions” or “CDRs” present in each antigen binding domain are short,non-contiguous sequences of amino acids that are specifically positionedto form the antigen binding domain as the antibody assumes its threedimensional configuration in an aqueous environment. The remainder ofthe amino acids in the antigen binding domains, referred to as“framework” regions, show less inter-molecular variability. Theframework regions largely adopt a β-sheet conformation and the CDRs formloops that connect, and in some cases form part of, the β-sheetstructure. Thus, framework regions act to form a scaffold that providesfor positioning the CDRs in correct orientation by inter-chain,non-covalent interactions. The antigen binding domain formed by thepositioned CDRs defines a surface complementary to the epitope on theimmunoreactive antigen. This complementary surface promotes thenon-covalent binding of the antibody to its cognate epitope. The aminoacids comprising the CDRs and the framework regions, respectively, canbe readily identified for any given heavy or light chain variable domainby one of ordinary skill in the art since they have been preciselydefined (see, “Sequences of Proteins of Immunological Interest,” Kabatet al. (1983) U.S. Department of Health and Human Services; and Chothiaand Lesk (1987) J. Mol. Biol., 196:901-917, which are incorporatedherein by reference in their entireties).

In the case where there are two or more definitions of a term that isused and/or accepted within the art, the definition of the term as usedherein is intended to include all such meanings unless explicitly statedto the contrary. A specific example is the use of the term“complementarity determining region” (“CDR”) to describe thenon-contiguous antigen combining sites found within the variable regionof both heavy and light chain polypeptides. This particular region hasbeen described by Kabat et al. (1983) U.S. Dept. of Health and HumanServices, “Sequences of Proteins of Immunological Interest” and byChothia and Lesk (1987) J. Mol. Biol. 196:901-917, which areincorporated herein by reference, where the definitions includeoverlapping or subsets of amino acid residues when compared against eachother. Nevertheless, application of either definition to refer to a CDRof an antibody or variants thereof is intended to be within the scope ofthe term as defined and used herein. Those skilled in the art canroutinely determine which residues comprise a particular CDR given thevariable region amino acid sequence of the antibody.

Kabat et al. also defined a numbering system for variable domainsequences that is applicable to any antibody. One of ordinary skill inthe art can unambigously assign this system of “Kabat numbering” to anyvariable domain sequence, without reliance on any experimental databeyond the sequence itself. As used herein, “Kabat numbering” refers tothe numbering system set forth by Kabat et al. (1983) U.S. Dept. ofHealth and Human Services, “Sequence of Proteins of ImmunologicalInterest.” Unless otherwise specified, references to the numbering ofspecific amino acid residue positions in an anti-factor XI monoclonalantibody or antigen-binding fragment, variant, or derivative thereof ofthe present invention are according to the Kabat numbering system.

Antibodies or antigen-binding fragments, variants, or derivativesthereof of the invention include, but are not limited to monoclonal,human, humanized, primatized, or chimeric antibodies, single chainantibodies, epitope-binding fragments, e.g., Fab, Fab′ and F(ab′)₂, Fd,Fvs, single-chain Fvs (scFv), single-chain antibodies, disulfide-linkedFvs (sdFv), fragments comprising either a V_(L) or V_(H) domain,fragments produced by a Fab expression library, and anti-idiotypic(anti-Id) antibodies (including, e.g., anti-Id antibodies to anti-factorXI monoclonal antibodies disclosed herein). ScFv molecules are known inthe art and are described, e.g., in U.S. Pat. No. 5,892,019.Immunoglobulin or antibody molecules of the invention can be of any type(e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3,IgG4, IgA1, and IgA2, etc.), or subclass of immunoglobulin molecule.

Antibody fragments, including single-chain antibodies, may comprise thevariable region(s) alone or in combination with the entirety or aportion of the following: hinge region, C_(H)1, C_(H)2, and C_(H)3domains. Also included in the invention are antigen-binding fragmentsalso comprising any combination of variable region(s) with a hingeregion, C_(H)1, C_(H)2, and C_(H)3 domains.

As used herein, “human” antibodies include antibodies having the aminoacid sequence of a human immunoglobulin and include antibodies isolatedfrom human immunoglobulin libraries or from animals transgenic for oneor more human immunoglobulins and that do not express endogenousimmunoglobulins, as described infra and, for example in, U.S. Pat. No.5,939,598 by Kucherlapati et al.

As used herein, the term “heavy chain portion” includes amino acidsequences derived from an immunoglobulin heavy chain. A polypeptidecomprising a heavy chain portion comprises at least one of: a C_(H)1domain, a hinge (e.g., upper, middle, and/or lower hinge region) domain,a C_(H)2 domain, a C_(H)3 domain, or a variant or fragment thereof. Forexample, a binding polypeptide for use in the invention may comprise apolypeptide chain comprising a C_(H)1 domain; a polypeptide chaincomprising a C_(H)1 domain, at least a portion of a hinge domain, and aC_(H)2 domain; a polypeptide chain comprising a C_(H)1 domain and aC_(H)3 domain; a polypeptide chain comprising a C_(H)1 domain, at leasta portion of a hinge domain, and a C_(H)3 domain, or a polypeptide chaincomprising a C_(H)1 domain, at least a portion of a hinge domain, aC_(H)2 domain, and a C_(H)3 domain. In another embodiment, a polypeptideof the invention comprises a polypeptide chain comprising a C_(H)3domain. Further, a binding polypeptide for use in the invention may lackat least a portion of a C_(H)2 domain (e.g., all or part of a C_(H)2domain). As set forth above, it will be understood by one of ordinaryskill in the art that these domains (e.g., the heavy chain portions) maybe modified such that they vary in amino acid sequence from thenaturally occurring immunoglobulin molecule.

Anti-factor XI monoclonal antibodies, or antigen-binding fragments,variants, or derivatives thereof disclosed herein may be described orspecified in terms of the epitope(s) or portion(s) of an antigen, e.g.,a target polypeptide (human factor XI, described in SEQ ID NO:1) thatthey recognize or specifically bind. The portion of a target polypeptidethat specifically interacts with the antigen binding domain of anantibody is an “epitope,” or an “antigenic determinant.” A targetpolypeptide may comprise a single epitope, but typically comprises atleast two epitopes, and can include any number of epitopes, depending onthe size, conformation, and type of antigen. Furthermore, it should benoted that an “epitope” on a target polypeptide may be or includenon-polypeptide elements, e.g., an epitope may include a carbohydrateside chain.

The minimum size of a peptide or polypeptide epitope for an antibody isthought to be about four to five amino acids. Peptide or polypeptideepitopes preferably contain at least seven, more preferably at leastnine and most preferably between at least about 15 to about 30 aminoacids. Since a CDR can recognize an antigenic peptide or polypeptide inits tertiary form, the amino acids comprising an epitope need not becontiguous, and in some cases, may not even be on the same peptidechain. In the present invention, peptide or polypeptide epitoperecognized by anti-factor XI monoclonal antibodies of the presentinvention is located on the heavy chain of human factor XI and containsa sequence of at least 4, at least 5, at least 6, at least 7, morepreferably at least 8, at least 9, at least 10, at least 15, at least20, at least 25, or between about 15 to about 30 contiguous ornon-contiguous amino acids of human factor XI (SEQ ID NO:1).

By “specifically binds,” it is generally meant that an antibody binds toan epitope via its antigen binding domain, and that the binding entailssome complementarity between the antigen binding domain and the epitope.According to this definition, an antibody is said to “specifically bind”to an epitope when it binds to that epitope, via its antigen bindingdomain more readily than it would bind to a random, unrelated epitope.The term “specificity” is used herein to qualify the relative affinityby which a certain antibody binds to a certain epitope. For example,antibody “A” may be deemed to have a higher specificity for a givenepitope than antibody “B,” or antibody “A” may be said to bind toepitope “C” with a higher specificity than it has for related epitope“D.”

By “preferentially binds,” it is meant that the antibody specificallybinds to an epitope more readily than it would bind to a related,similar, homologous, or analogous epitope. Thus, an antibody that“preferentially binds” to a given epitope would more likely bind to thatepitope than to a related epitope, even though such an antibody maycross-react with the related epitope.

By way of non-limiting example, antibody may be considered to bind afirst epitope preferentially if it binds said first epitope with adissociation constant (K_(D)) that is less than the antibody's K_(D) forthe second epitope. In another non-limiting example, an antibody may beconsidered to bind a first antigen preferentially if it binds the firstepitope with an affinity that is at least one order of magnitude lessthan the antibody's K_(D) for the second epitope. In anothernon-limiting example, an antibody may be considered to bind a firstepitope preferentially if it binds the first epitope with an affinitythat is at least two orders of magnitude less than the antibody's K_(D)for the second epitope.

In another non-limiting example, an antibody may be considered to bind afirst epitope preferentially if it binds the first epitope with an offrate (k(off)) that is less than the antibody's k(off) for the secondepitope. In another non-limiting example, an antibody may be consideredto bind a first epitope preferentially if it binds the first epitopewith an affinity that is at least one order of magnitude less than theantibody's k(off) for the second epitope. In another non-limitingexample, an antibody may be considered to bind a first epitopepreferentially if it binds the first epitope with an affinity that is atleast two orders of magnitude less than the antibody's k(off) for thesecond epitope.

An antibody or antigen-binding fragment, variant, or derivativedisclosed herein may be said to bind a target polypeptide disclosedherein or a fragment or variant thereof with an off rate (k(off)) ofless than or equal to 5×10⁻² sec⁻¹, 10⁻² sec⁻¹, 5×10⁻³ sec⁻¹, or 10⁻³sec⁻¹. More preferably, an antibody of the invention may be said to binda target polypeptide disclosed herein or a fragment or variant thereofwith an off rate (k(off)) less than or equal to 5×10⁻⁴ sec⁻¹, 10⁻⁴sec⁻¹, 5×10⁻⁵ sec⁻¹, or 10⁻⁵ sec⁻¹, 5×10⁻⁶ sec⁻¹, 10⁻⁶ sec⁻¹, 5×10⁻⁷sec⁻¹, or 10⁻⁷ sec⁻¹.

An antibody or antigen-binding fragment, variant, or derivative thereofdisclosed herein may be said to bind a target polypeptide disclosedherein or a fragment or variant thereof with an on rate (k(on)) ofgreater than or equal to 10³ M⁻¹ sec⁻¹, 5×10³ M⁻¹ sec⁻¹, 10⁴ M⁻¹ sec⁻¹,or 5×10⁴ M⁻¹ sec⁻¹. More preferably, an antibody of the invention may besaid to bind a target polypeptide disclosed herein or a fragment orvariant thereof with an on rate (k(on)) greater than or equal to 10⁵ M⁻¹sec⁻¹, 5×10⁵ M⁻¹ sec⁻¹, 10⁶ M⁻¹ sec⁻¹, 5×10⁶ M⁻¹ sec⁻¹, or 10⁷ M⁻¹sec⁻¹.

An antibody is said to competitively inhibit binding of a referenceantibody to a given epitope if it preferentially binds to that epitopeto the extent that it blocks, to some degree, binding of the referenceantibody to the epitope. Competitive inhibition may be determined by anymethod known in the art, for example, competition ELISA assays. Anantibody may be said to competitively inhibit binding of the referenceantibody to a given epitope by at least 90%, at least 80%, at least 70%,at least 60%, or at least 50%.

As used herein, the term “affinity” refers to a measure of the strengthof the binding of an individual epitope with the CDR of animmunoglobulin molecule. See, e.g., Harlow et al. (1988) Antibodies: ALaboratory Manual (Cold Spring Harbor Laboratory Press, 2nd ed.) pages27-28. As used herein, the term “avidity” refers to the overallstability of the complex between a population of immunoglobulins and anantigen, that is, the functional combining strength of an immunoglobulinmixture with the antigen. See, e.g., Harlow at pages 29-34. Avidity isrelated to both the affinity of individual immunoglobulin molecules inthe population with specific epitopes, and also the valencies of theimmunoglobulins and the antigen. For example, the interaction between abivalent monoclonal antibody and an antigen with a highly repeatingepitope structure, such as a polymer, would be one of high avidity.

Anti-factor XI monoclonal antibodies or antigen-binding fragments,variants or derivatives thereof of the invention may also be describedor specified in terms of their binding affinity to a polypeptide of theinvention. Preferred binding affinities include those with adissociation constant or Kd less than 5×10⁻² M, 10⁻² M, 5×10⁻³ M, 10⁻³M, 5×10⁻⁴ M, 10⁻⁴ M, 5×10⁻⁵ M, 10⁻⁵ M, 5×10⁻⁶ M, 10⁻⁶ M, 5×10⁻⁷ M, 10⁻⁷M, 5×10⁻⁸ M, 10⁻⁸ M, 5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M,10⁻¹¹ M, 5×10⁻¹² M, 10⁻¹² M, 5×10⁻¹³ M, 10⁻¹³ M, 5×10⁻¹⁴ M, 10⁻¹⁴ M,5×10⁻¹⁵ M, or 10⁻¹⁵ M.

As used herein, the term “chimeric antibody” will be held to mean anyantibody wherein the immunoreactive region or site is obtained orderived from a first species and the constant region (which may beintact, partial or modified in accordance with the instant invention) isobtained from a second species. In preferred embodiments the targetbinding region or site will be from a non-human source (e.g., mouse orprimate) and the constant region is human.

As used herein, the term “engineered antibody” refers to an antibody inwhich the variable domain in either the heavy or light chain or both isaltered by at least partial replacement of one or more CDRs from anantibody of known specificity and, if necessary, by partial frameworkregion replacement and sequence changing. Although the CDRs may bederived from an antibody of the same class or even subclass as theantibody from which the framework regions are derived, it is envisagedthat the CDRs will be derived from an antibody of different class andpreferably from an antibody from a different species. An engineeredantibody in which one or more “donor” CDRs from a non-human antibody ofknown specificity is grafted into a human heavy or light chain frameworkregion is referred to herein as a “humanized antibody.” It may not benecessary to replace all of the CDRs with the complete CDRs from thedonor variable domain to transfer the antigen binding capacity of onevariable domain to another. Rather, it may only be necessary to transferthose residues that are necessary to maintain the activity of the targetbinding site.

It is further recognized that the framework regions within the variabledomain in a heavy or light chain, or both, of a humanized antibody maycomprise solely residues of human origin, in which case these frameworkregions of the humanized antibody are referred to as “fully humanframework regions.” Alternatively, one or more residues of the frameworkregion(s) of the donor variable domain can be engineered within thecorresponding position of the human framework region(s) of a variabledomain in a heavy or light chain, or both, of a humanized antibody ifnecessary to maintain proper binding or to enhance binding to the humanfactor XI antigen. A human framework region that has been engineered inthis manner would thus comprise a mixture of human and donor frameworkresidues, and is referred to herein as a “partially human frameworkregion.” Given the explanations set forth in, e.g., U.S. Pat. Nos.5,585,089, 5,693,761, 5,693,762, and 6,180,370 it will be well withinthe competence of those skilled in the art, either by carrying outroutine experimentation or by trial and error testing to obtain afunctional engineered or humanized antibody.

For example, humanization of an anti-factor XI monoclonal antibody canbe essentially performed following the method of Winter and co-workers(Jones et al. (1986) Nature 321:522-525; Riechmann et al. (1988) Nature332:323-327; Verhoeyen et al. (1988) Science 239:1534-1536), bysubstituting rodent or mutant rodent CDRs or CDR sequences for thecorresponding sequences of a human anti-factor XI monoclonal antibody.See also U.S. Pat. Nos. 5,225,539; 5,585,089; 5,693,761; 5,693,762;5,859,205; herein incorporated by reference. The resulting humanizedanti-factor XI monoclonal antibody would comprise at least one rodent ormutant rodent CDR within the fully human framework regions of thevariable domain of the heavy and/or light chain of the humanizedantibody. In some instances, residues within the framework regions ofone or more variable domains of the humanized anti-factor XI monoclonalantibody are replaced by corresponding non-human (for example, rodent)residues (see, for example, U.S. Pat. Nos. 5,585,089; 5,693,761;5,693,762; and 6,180,370), in which case the resulting humanizedanti-factor XI monoclonal antibody would comprise partially humanframework regions within the variable domain of the heavy and/or lightchain.

Furthermore, humanized antibodies may comprise residues that are notfound in the recipient antibody or in the donor antibody. Thesemodifications are made to further refine antibody performance (e.g., toobtain desired affinity). In general, the humanized antibody willcomprise substantially all of at least one, and typically two, variabledomains, in which all or substantially all of the CDRs correspond tothose of a non-human immunoglobulin and all or substantially all of theframework regions are those of a human immunoglobulin sequence. Thehumanized antibody optionally also will comprise at least a portion ofan immunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details see Jones et al. (1986) Nature331:522-525; Riechmann et al. (1988) Nature 332:323-329; and Presta(1992) Curr. Op. Struct. Biol. 2:593-596; herein incorporated byreference. Accordingly, such “humanized” antibodies may includeantibodies wherein substantially less than an intact human variabledomain has been substituted by the corresponding sequence from anon-human species. In practice, humanized antibodies are typically humanantibodies in which some CDR residues and possibly some frameworkresidues are substituted by residues from analogous sites in rodentantibodies. See, for example, U.S. Pat. Nos. 5,225,539; 5,585,089;5,693,761; 5,693,762; 5,859,205. See also U.S. Pat. No. 6,180,370, andInternational Publication No. WO 01/27160, where humanized antibodiesand techniques for producing humanized antibodies having improvedaffinity for a predetermined antigen are disclosed.

As used herein the term “properly folded polypeptide” includespolypeptides (e.g., anti-factor XI monoclonal antibodies) in which allof the functional domains comprising the polypeptide are distinctlyactive. As used herein, the term “improperly folded polypeptide”includes polypeptides in which at least one of the functional domains ofthe polypeptide is not active. In one embodiment, a properly foldedpolypeptide comprises polypeptide chains linked by at least onedisulfide bond and, conversely, an improperly folded polypeptidecomprises polypeptide chains not linked by at least one disulfide bond.

As used herein the term “engineered” includes manipulation of nucleicacid or polypeptide molecules by synthetic means (e.g., by recombinanttechniques, in vitro peptide synthesis, by enzymatic or chemicalcoupling of peptides or some combination of these techniques).

The term “expression” as used herein refers to a process by which a geneproduces a biochemical, for example, a polypeptide. The process includesany manifestation of the functional presence of the gene within the cellincluding, without limitation, gene knockdown as well as both transientexpression and stable expression. It includes without limitationtranscription of the gene into messenger RNA (mRNA), and the translationof such mRNA into polypeptide(s). If the final desired product is abiochemical, expression includes the creation of that biochemical andany precursors. Expression of a gene produces a “gene product.” As usedherein, a gene product can be either a nucleic acid, e.g., a messengerRNA produced by transcription of a gene, or a polypeptide which istranslated from a transcript. Gene products described herein furtherinclude nucleic acids with post transcriptional modifications, e.g.,polyadenylation, or polypeptides with post translational modifications,e.g., methylation, glycosylation, the addition of lipids, associationwith other protein subunits, proteolytic cleavage, and the like.

As used herein, the terms “treat” or “treatment” refer to boththerapeutic treatment and prophylactic or preventative measures, whereinthe object is to prevent or slow down (lessen) an undesiredphysiological change or disorder, such as the progression of a diseasestate. Beneficial or desired clinical results include, but are notlimited to, alleviation of symptoms, diminishment of extent of disease,stabilized (i.e., not worsening) state of disease, delay or slowing ofdisease progression, amelioration or palliation of the disease state,and remission (whether partial or total), whether detectable orundetectable. “Treatment” can also mean prolonging survival as comparedto expected survival if not receiving treatment. Those in need oftreatment include those already with the condition or disorder as wellas those prone to have the condition or disorder or those in which thecondition or disorder is to be prevented.

By “subject” or “individual” or “animal” or “patient” or “mammal,” ismeant any subject, particularly a mammalian subject, for whom diagnosis,prognosis, or therapy is desired. Mammalian subjects include humans,non-human primates, domestic animals, farm animals, and zoo, sports, orpet animals such as dogs, cats, guinea pigs, rabbits, rats, mice,horses, cattle, cows, and the like.

As used herein, phrases such as “a subject that would benefit fromadministration of an anti-factor XI monoclonal antibody” and “an animalin need of treatment” includes subjects, such as mammalian subjects,that would benefit from administration of an anti-factor XI monoclonalantibody used, e.g., for inhibiting thrombosis with an anti-factor XImonoclonal antibody.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology, transgenic biology, microbiology, recombinant DNA,and immunology, which are within the skill of the art. Such techniquesare explained fully in the literature. See, for example, Sambrook etal., ed. (1989) Molecular Cloning A Laboratory Manual (2nd ed.; ColdSpring Harbor Laboratory Press); Sambrook et al., ed. (1992) MolecularCloning: A Laboratory Manual, (Cold Springs Harbor Laboratory, NY); D.N. Glover ed., (1985) DNA Cloning, Volumes I and II; Gait, ed. (1984)Oligonucleotide Synthesis; Mullis et al. U.S. Pat. No. 4,683,195; Hamesand Higgins, eds. (1984) Nucleic Acid Hybridization; Hames and Higgins,eds. (1984) Transcription And Translation; Freshney (1987) Culture OfAnimal Cells (Alan R. Liss, Inc.); Immobilized Cells And Enzymes (IRLPress) (1986); Perbal (1984) A Practical Guide To Molecular Cloning; thetreatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Miller andCalos eds. (1987) Gene Transfer Vectors For Mammalian Cells, (ColdSpring Harbor Laboratory); Wu et al., eds., Methods In Enzymology, Vols.154 and 155; Mayer and Walker, eds. (1987) Immunochemical Methods InCell And Molecular Biology (Academic Press, London); Weir and Blackwell,eds., (1986) Handbook Of Experimental Immunology, Volumes I-IV;Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., (1986); and in Ausubel et al. (1989) CurrentProtocols in Molecular Biology (John Wiley and Sons, Baltimore, Md.).

General principles of antibody engineering are set forth in Borrebaeck,ed. (1995) Antibody Engineering (2nd ed.; Oxford Univ. Press). Generalprinciples of protein engineering are set forth in Rickwood et al., eds.(1995) Protein Engineering, A Practical Approach (IRL Press at OxfordUniv. Press, Oxford, Eng.). General principles of antibodies andantibody-hapten binding are set forth in: Nisonoff (1984) MolecularImmunology (2nd ed.; Sinauer Associates, Sunderland, Mass.); and Steward(1984) Antibodies, Their Structure and Function (Chapman and Hall, NewYork, N.Y.). Additionally, standard methods in immunology known in theart and not specifically described are generally followed as in CurrentProtocols in Immunology, John Wiley & Sons, New York; Stites et al.,eds. (1994) Basic and Clinical Immunology (8th ed; Appleton & Lange,Norwalk, Conn.) and Mishell and Shiigi (eds) (1980) Selected Methods inCellular Immunology (W.H. Freeman and Co., NY).

Standard reference works setting forth general principles of immunologyinclude Current Protocols in Immunology, John Wiley & Sons, New York;Klein (1982) J. Immunology: The Science of Self-Nonself Discrimination(John Wiley & Sons, NY); Kennett et al., eds. (1980) MonoclonalAntibodies, Hybridoma: A New Dimension in Biological Analyses (PlenumPress, NY); Campbell (1984) “Monoclonal Antibody Technology” inLaboratory Techniques in Biochemistry and Molecular Biology, ed. Burdenet al., (Elsevere, Amsterdam); Goldsby et al., eds. (2000) KubyImmunnology (4th ed.; H. Freemand & Co.); Roitt et al. (2001) Immunology(6th ed.; London: Mosby); Abbas et al. (2005) Cellular and MolecularImmunology (5th ed.; Elsevier Health Sciences Division); Kontermann andDubel (2001) Antibody Engineering (Springer Verlan); Sambrook andRussell (2001) Molecular Cloning: A Laboratory Manual (Cold SpringHarbor Press); Lewin (2003) Genes VIII (Prentice Hall 2003); Harlow andLane (1988) Antibodies: A Laboratory Manual (Cold Spring Harbor Press);Dieffenbach and Dveksler (2003) PCR Primer (Cold Spring Harbor Press).

III. Human Factor XI

Human Factor XI is a two-chain glycoprotein with a molecular weight ofapproximately 160,000 daltons. The two chains are identical disulfidebonded polypeptides with molecular weights of approximately 80,000daltons. Factor XI is activated to factor XIa by Factor XIIa. The aminoacid sequence of human factor XI has been determined (see, e.g.,Fujikawa et al. (1986) Biochemistry 25:2417-2424) and is provided as SEQID NO:1. In humans, the gene for FXI is located at the distal end ofchromosome 4 (4q35.2) and contains 15 exons spread over ˜25 kb ofgenomic DNA (Asaki et al. (1987) Biochemistry 26:7221-7228; Kato et al.(1989) Cytogenet. Cell Genet. 52:77).

The cleavage site for the activation of factor XI by factor XIIa is aninternal peptide bond between Arg-369 and Ile-370 in each polypeptidechain (Fujikawa et al. (1986) Biochemistry 25:2417-2424). Each heavychain of factor XIa (369 amino acids) contains four tandem repeats of90-91 amino acids called apple domains (designated A1-A4) plus a shortconnecting peptide (Fujikawa et al. (1986) Biochemistry 25:2417-2424;Sun et al. (1999) J. Biol. Chem. 274:36373-36378). The light chains offactor XIa (each 238 amino acids) contain the catalytic portion of theenzyme with sequences that are typical of the trypsin family of serineproteases (Fujikawa et al. (1986) Biochemistry 25:2417-2424). XIaproteolytically cleaves its substrate, factor IX, in an interactionrequiring the factor XI A3 domain (Sun, Y., and Gailani, D. (1996) J.Biol. Chem. 271, 29023-29028).

IV. Anti-Factor XI Monoclonal Antibodies

In one embodiment, the present invention is directed to anti-factor XImonoclonal antibodies, including antigen-binding fragments, variants, orderivatives thereof. As used herein, the term “anti-factor XI monoclonalantibody” is an antibody that specifically recognizes human factor XI,particularly an epitope in the A3 domain of the heavy chain of human FXI(positions 182 to 265 of SEQ ID NO:1). In one embodiment, the epitopecomprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or moreamino acids of the A3 domain of human FXI. In another embodiment, theepitope is selected from the group consisting of: a) amino acids 183 to197 of SEQ ID NO:1; b) amino acids 203 to 204 of SEQ ID NO:1; c) aminoacids 234 to 236 of SEQ NO:1; d) amino acids 241 to 243 of SEQ ID NO:1;e) amino acids 252 to 254 of SEQ ID NO:1; and f) amino acids 258 to 260of SEQ ID NO:1. In particular embodiments, the epitope is selected fromthe group consisting of: a) amino acids 183 to 197 of SEQ ID NO:1; b)amino acids 252 to 254 of SEQ ID NO:1; and c) amino acids 258 to 260 ofSEQ ID NO:1.

In another embodiment, the anti-factor XI monoclonal antibodies of theinvention have binding specificity to an antigen that is a peptidecomprising 2 or more amino acids out of the A3 domain of the heavy chainof human FXI (positions 182 to 265 of SEQ ID NO:1). In yet anotherembodiment, the anti-factor XI monoclonal antibodies of the inventionhave binding specificity to an antigen that is a peptide comprising anamino acid sequence selected from the group consisting of: a) aminoacids 183 to 197 of SEQ ID NO:1; b) amino acids 203 to 204 of SEQ IDNO:1; c) amino acids 234 to 236 of SEQ ID NO:1; d) amino acids 241 to243 of SEQ ID NO:1; e) amino acids 252 to 254 of SEQ ID NO:1; and f)amino acids 258 to 260 of SEQ ID NO:1. In particular embodiments, theanti-factor XI monoclonal antibodies of the invention have bindingspecificity to an antigen that is a peptide comprising an amino acidsequence selected from the group consisting of: a) amino acids 183 to197 of SEQ ID NO:1; b) amino acids 252 to 254 of SEQ ID NO:1; and c)amino acids 258 to 260 of SEQ ID NO:1.

The anti-factor XI monoclonal antibodies of the invention, includingantigen-binding fragments, variants, or derivatives thereof, arebiologically active. The term “biologically active” as used hereinrefers to one or more of the following physiological activitiesassociated with the antithrombotic activity of the anti-factor XImonoclonal antibodies of the invention: 1) inhibition of the coagulationactivity of activated factor XI; 2) prevention of the activation offactor XI; and 3) acceleration of the clearance of factor XI from thecirculatory system of a subject.

In one embodiment, the term “biologically active” refers to inhibitingthe coagulation activity of activated human factor XI. The phrase“inhibit the coagulation activity” as used herein refers to decreasingthe ability of activated factor XI to produce blood clot formation.Methods for determining whether the coagulation activity has beeninhibited include the use of assays for measuring clot strength and/orthe length of time before clot formation in plasma or whole bloodsamples. Accordingly, inhibiting coagulation activity as used hereinrefers to at least partially reversing the effect of an coagulant,including at least 5% reversal, at least 10% reversal, at least 20%reversal, at least 30% reversal, at least 40% reversal, at least 50%reversal, at least 60% reversal, at least 70% reversal, at least 80%reversal, at least 90% reversal, and up to and including 100% reversal.The term “reversal” as used herein refers to a lengthening of the timeto onset of clot formation or a decrease in clot strength. Assays formeasuring the onset of clot formation and clot strength are well knownin the art and include activated partial thromboplastin time (APTT),thromboelastography (TEG®), and continuous monitoring of thrombingeneration using the Thrombinoscope® system (see, for example, Banez etal. (1980) Am. J. Clin. Pathol., 74:569-574; van den Besselaar et al.(1990) Thromb. Haemost., 63:16-23; Kawasaki et al. (2004) Anesthesia &Analgesia, 99:1440-1444; Hemker et al. (2003) Pathophysiology ofHaemostasis & Thrombosis, 33:4-15).

In one embodiment, the monoclonal antibody of the invention is producedby hybridoma cell line 1A6.

The present invention also relates to humanized anti-factor XIantibodies that bind to human factor XI. In some embodiments, theanti-factor XI antibodies of the invention comprise at least oneoptimized complementarity-determining region (CDR). By “optimized CDR”is intended that the CDR has been modified and optimized sequencesselected based on the sustained or improved binding affinity and/oranti-factor XI activity that is imparted to an anti-factor XI antibodycomprising the optimized CDR. “Anti-factor XI activity” or “factor XIblocking activity” can include one or more of the followingphysiological activities, as described elsewhere herein: 1) inhibitionof the coagulation activity of activated factor XI; 2) prevention of theactivation of factor XI; and 3) acceleration of the clearance of factorXI from the circulatory system of a subject.

The modifications involve replacement of amino acid residues within theCDR such that an anti-factor XI antibody retains specificity for thefactor XI antigen and has improved binding affinity and/or improvedanti-factor XI activity. Anti-factor XI activity of an anti-factor XIantibody of the invention is improved as compared to aXIMab (1A6) in afunctional assay as described herein. The novel anti-factor XIantibodies of the invention and suitable antigen-binding fragments,variants, and derivatives thereof also exhibit anti-factor XI activitythat is at least similar to that exhibited by aXIMab (1A6), as measuredin standard assays. The optimized CDRs of the invention are utilized inV_(H) and V_(L) domains of the heavy and light chains, respectively, ofanti-factor XI antibodies. Exemplary anti-factor XI antibodies of theinvention comprise a V_(H) domain selected from the group consisting ofSEQ ID NO:3 and 7, and/or a V_(L) domain selected from the groupconsisting of SEQ ID NO:5 and 9.

The anti-factor XI antibodies of the invention comprise at least oneoptimized complementarity-determining region (CDR). By “optimized CDR”is intended that the CDR has been modified and optimized sequencesselected based on the improved binding affinity and/or improved CDCactivity that is imparted to an anti-factor XI antibody comprising theoptimized CDR. The modifications involve replacement of amino acidresidues within the CDR such that an anti-factor XI antibody retainsspecificity for the factor XI antigen and has improved or sustainedbinding affinity and/or anti-factor XI activity. Anti-factor XI activityof an anti-factor XI antibody of the invention is improved or sustainedas compared to aXIMab (1A6) in a functional assay as described herein.The optimized CDRs of the invention are utilized in V_(H) and V_(L)domains of the heavy and light chains, respectively, of anti-humanfactor XI antibodies. Exemplary anti-factor XI antibodies of theinvention comprise a V_(H) domain selected from the group consisting ofSEQ ID NO:3 and 7, and/or a V_(L) domain selected from the groupconsisting of SEQ ID NO:5 and 9.

In some embodiments, the anti-factor XI antibodies of the inventioncomprise optimized CDRs. That is, the anti-factor XI antibodies of theinvention comprise at least one optimized CDR amino acid sequenceselected from the group consisting of SEQ ID NO:10-15 or amino acidsequences having at least about 65%, about 70%, about 75%, about 80%,about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about99%, or 100% sequence identity to a sequence selected from the groupconsisting of SEQ ID NO:10-15. That is, the optimized CDRs comprise thesequences set forth in SEQ ID NO:10-15 and the sequences of SEQ IDNO:10-15 having at least one, two, three, four, or five amino acidsubstitutions, depending upon the CDR involved.

Thus, in some embodiments, the anti-factor XI antibodies of theinvention comprise a V_(H) domain having at least one optimized CDRselected from the group consisting of:

a) a CDR comprising the amino acid sequence set forth in SEQ ID NO:10,11, or 12; and

b) a CDR comprising an amino acid sequence having at least 85% sequenceidentity to the amino acid sequence set forth in SEQ ID NO:10, 11, or12.

In other embodiments, the anti-factor XI antibodies of the inventioncomprise a V_(L) domain having at least one optimized CDR selected fromthe group consisting of:

a) a CDR comprising the amino acid sequence set forth in SEQ ID NO:13,14, or 15; and

b) a CDR comprising an amino acid sequence having at least 85% sequenceidentity to the amino acid sequence set forth in SEQ ID NO:13, 14, or15.

In other embodiments, the anti-factor XI antibodies of the inventioncomprise a V_(H) domain and a V_(L) domain, wherein said V_(H) domainhas at least one optimized CDR comprising the amino acid sequence setforth in SEQ ID NO:10, 11, or 12, and wherein said V_(L) domain has atleast one optimized CDR comprising the amino acid sequence set forth inSEQ ID NO:13, 14, or 15.

In some embodiments, the anti-factor XI antibodies comprising at leastone of the optimized CDRs of the invention are IgG kappaimmunoglobulins. In such embodiments, the IgG kappa immunoglobulin cancomprise a human IgG1, IgG2, IgG3, or IgG4 constant region within aheavy chain of the immunoglobulin and a human kappa constant regionwithin a light chain of the immunoglobulin. In particular embodiments,the IgG kappa immunoglobulin comprises fully or partially humanframework regions within the variable domain of the heavy chain andwithin the variable domain of the light chain. In other embodiments, theIgG kappa immunoglobulin comprises murine framework regions within thevariable domain of the heavy chain and within the variable domain of thelight chain.

In further embodiments of the invention, the anti-factor XI antibodiesof the invention comprise a V_(H) domain having an amino acid sequenceset forth in SEQ ID NO:3 or 7 and/or a V_(L) domain having an amino acidsequence set forth in SEQ ID NO:5 and 9, or amino acid sequences havingat least about 80%, 85%, about 90%, about 91%, about 92%, about 93%,about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or 100%sequence identity to a sequence set forth in SEQ ID NO:3, 5, 7, and 9.

In yet other embodiments of the invention, the anti-factor XI antibodiesof the invention comprise a V_(H) domain, where the V_(H) domain isselected from the group consisting of:

a) a V_(H) domain comprising an amino acid sequence set forth in SEQ IDNO:3 or 7; and

b) a V_(H) domain comprising an amino acid sequence having at least 85%sequence identity to an amino acid sequence set forth in SEQ ID NO:3 or7.

In another embodiment of the invention, the anti-factor XI antibodies ofthe invention comprise a V_(L) domain, where the V_(L) domain isselected from the group consisting of:

a) a V_(L) domain comprising an amino acid sequence set forth in SEQ IDNO:5 or 9; and

b) a V_(L) domain comprising an amino acid sequence having at least 85%sequence identity to an amino acid sequence set forth in SEQ ID NO:5 or9.

Methods for measuring anti-factor XI antibody binding specificityinclude, but are not limited to, standard competitive binding assays,assays for monitoring immunoglobulin secretion by T cells or B cells, Tcell proliferation assays, apoptosis assays, ELISA assays, and the like.See, for example, such assays disclosed in WO 93/14125; Shi et al.(2000) Immunity 13:633-642; Kumanogoh et al. (2002) J Immunol169:1175-1181; Watanabe et al. (2001) J Immunol 167:4321-4328; Wang etal. (2001) Blood 97:3498-3504; and Giraudon et al. (2004) J Immunol172(2):1246-1255, all of which are herein incorporated by reference.

Suitable biologically active variants of the anti-factor XI antibodiescan be used in the methods of the present invention. Such variants willretain the desired binding properties of the parent anti-factor XIantibody. Methods for making antibody variants are generally availablein the art. For example, amino acid sequence variants of an anti-factorXI antibody, an antibody region, or an antibody variable domain of aheavy or light chain, can be prepared by mutations in the cloned DNAsequence encoding the amino acid sequence of interest. Methods formutagenesis and nucleotide sequence alterations are well known in theart. See, for example, Walker and Gaastra, eds. (1983) Techniques inMolecular Biology (MacMillan Publishing Company, New York); Kunkel(1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987)Methods Enzymol. 154:367-382; Sambrook et al. (1989) Molecular Cloning:A Laboratory Manual (Cold Spring Harbor, New York); U.S. Pat. No.4,873,192; and the references cited therein; herein incorporated byreference. Guidance as to appropriate amino acid substitutions that donot affect biological activity of the polypeptide of interest may befound in the model of Dayhoff et al. (1978) in Atlas of Protein Sequenceand Structure (Natl. Biomed. Res. Found, Washington, D.C.), pp. 345-352,herein incorporated by reference in its entirety. The model of Dayhoffet al. uses the Point Accepted Mutation (PAM) amino acid similaritymatrix (PAM 250 matrix) to determine suitable conservative amino acidsubstitutions. Conservative substitutions, such as exchanging one aminoacid with another having similar properties, may be preferred. Examplesof conservative amino acid substitutions as taught by the PAM 250 matrixof the Dayhoff et al. model include, but are not limited to, Gly⇔Ala,Val⇔Ile⇔Leu, Asp⇔Glu, Lys⇔Arg, Asn⇔Gln, and Phe⇔Trp⇔Tyr.

In constructing variants of the anti-factor XI antibody polypeptides ofinterest, modifications are made such that variants continue to possessthe desired properties, i.e., being capable of specifically binding to ahuman factor XI antigen expressed on the surface of or secreted by ahuman cell, and having factor XI blocking activity, as described herein.Obviously, any mutations made in the DNA encoding the variantpolypeptide must not place the sequence out of reading frame andpreferably will not create complementary regions that could producesecondary mRNA structure. See EP Patent Application Publication No.75,444.

In addition, the constant region of an anti-factor XI antibody can bemutated to alter effector function in a number of ways. For example, seeU.S. Pat. No. 6,737,056B1 and U.S. Patent Application Publication No.2004/0132101A1, which disclose Fc mutations that optimize antibodybinding to Fc receptors.

Preferably, variants of a reference anti-factor XI antibody have aminoacid sequences that have at least about 80%, about 85%, about 88%, about90%, about 91%, about 92%, about 93%, about 94%, or about 95% sequenceidentity to the amino acid sequence for the reference anti-factor XIantibody molecule or to a shorter portion of the reference antibodymolecule. More preferably, the molecules share at least about 96%, about97%, about 98%, or about 99% sequence identity. When discussed herein,whether any particular polypeptide, including the constant regions,CDRs, V_(H) domains, and V_(L) domains disclosed herein, is at leastabout 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%,about 98%, about 99%, or even about 100% identical to anotherpolypeptide can be determined using methods and computerprograms/software known in the art such as, but not limited to, theBESTFIT program (Wisconsin Sequence Analysis Package, Version 8 forUnix, Genetics Computer Group, University Research Park, 575 ScienceDrive, Madison, Wis. 53711). BESTFIT uses the local homology algorithmof Smith and Waterman (1981) Adv. Appl. Math. 2:482-489, to find thebest segment of homology between two sequences. When using BESTFIT orany other sequence alignment program to determine whether a particularsequence is, for example, 95% identical to a reference sequenceaccording to the present invention, the parameters are set, of course,such that the percentage of identity is calculated over the full lengthof the reference polypeptide sequence and that gaps in homology of up to5% of the total number of amino acids in the reference sequence areallowed.

For purposes of the present invention, percent sequence identity isdetermined using the Smith-Waterman homology search algorithm using anaffine gap search with a gap open penalty of 12 and a gap extensionpenalty of 2, BLOSUM matrix of 62. The Smith-Waterman homology searchalgorithm is taught in Smith and Waterman (1981) Adv. Appl. Math.2:482-489. A variant may, for example, differ from the referenceanti-factor XI antibody by as few as 1 to 15 amino acid residues, as fewas 1 to 10 amino acid residues, such as 6-10, as few as 5, as few as 4,3, 2, or even 1 amino acid residue.

With respect to optimal alignment of two amino acid sequences, thecontiguous segment of the variant amino acid sequence may haveadditional amino acid residues or deleted amino acid residues withrespect to the reference amino acid sequence. The contiguous segmentused for comparison to the reference amino acid sequence will include atleast 20 contiguous amino acid residues, and may be 30, 40, 50, or moreamino acid residues. Corrections for sequence identity associated withconservative residue substitutions or gaps can be made (seeSmith-Waterman homology search algorithm).

When any two polypeptide sequences are optimally aligned for comparison,it is recognized that residues appearing opposite of one another withinthe alignment occupy positions within their respective polypeptides thatcorrespond to one another. Such positions are referred to herein as“corresponding positions” and the residues residing at correspondingpositions are referred to as “corresponding residues” or residues that“correspond” to one another. Thus, for example, where a polypeptide ofinterest is optimally aligned to a reference polypeptide sequencehaving, for example, 10 residues, the residue within the polypeptide ofinterest appearing opposite residue 5 of the reference sequence isreferred to as the “residue at the position corresponding to residue 5”of the reference sequence.

The precise chemical structure of a polypeptide capable of specificallybinding factor XI and retaining the desired factor XI blocking activitydepends on a number of factors. As ionizable amino and carboxyl groupsare present in the molecule, a particular polypeptide may be obtained asan acidic or basic salt, or in neutral form. All such preparations thatretain their biological activity when placed in suitable environmentalconditions are included in the definition of anti-factor XI antibodiesas used herein. Further, the primary amino acid sequence of thepolypeptide may be augmented by derivatization using sugar moieties(glycosylation) or by other supplementary molecules such as lipids,phosphate, acetyl groups and the like. It may also be augmented byconjugation with saccharides. Certain aspects of such augmentation areaccomplished through post-translational processing systems of theproducing host; other such modifications may be introduced in vitro. Inany event, such modifications are included in the definition of ananti-factor XI antibody used herein so long as the desired properties ofthe anti-factor XI antibody are not destroyed. It is expected that suchmodifications may quantitatively or qualitatively affect the activity,either by enhancing or diminishing the activity of the polypeptide, inthe various assays. Further, individual amino acid residues in the chainmay be modified by oxidation, reduction, or other derivatization, andthe polypeptide may be cleaved to obtain fragments that retain activity.Such alterations that do not destroy the desired properties (i.e.,binding specificity for factor XI and factor XI blocking activity) donot remove the polypeptide sequence from the definition of anti-factorXI antibodies of interest as used herein.

The art provides substantial guidance regarding the preparation and useof polypeptide variants. In preparing the anti-factor XI antibodyvariants, one of skill in the art can readily determine whichmodifications to the native protein nucleotide or amino acid sequencewill result in a variant that is suitable for use as a therapeuticallyactive component of a pharmaceutical composition used in the methods ofthe present invention.

In certain anti-factor XI antibodies, the Fc portion may be mutated todecrease effector function using techniques known in the art. Forexample, the deletion or inactivation (through point mutations or othermeans) of a constant region domain may reduce FC receptor binding of thecirculating modified antibody thereby increasing tumor localization. Inother cases it may be that constant region modifications consistent withthe instant invention moderate complement binding and thus reduce theserum half life and nonspecific association of a conjugated cytotoxin.Yet other modifications of the constant region may be used to modifydisulfide linkages or oligosaccharide moieties that allow for enhancedlocalization due to increased antigen specificity or antibodyflexibility. The resulting physiological profile, bioavailability andother biochemical effects of the modifications, such as tumorlocalization, biodistribution and serum half-life, may easily bemeasured and quantified using well known immunological techniqueswithout undue experimentation.

Anti-factor XI antibodies of the invention also include derivatives thatare modified, e.g., by the covalent attachment of any type of moleculeto the antibody such that covalent attachment does not prevent theantibody from specifically binding to its cognate epitope. For example,but not by way of limitation, the antibody derivatives includeantibodies that have been modified, e.g., by glycosylation, acetylation,pegylation, phosphorylation, amidation, derivatization by knownprotecting/blocking groups, proteolytic cleavage, linkage to a cellularligand or other protein, etc. Any of numerous chemical modifications maybe carried out by known techniques, including, but not limited tospecific chemical cleavage, acetylation, formylation, etc. Additionally,the derivative may contain one or more non-classical amino acids.

A “conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a side chain witha similar charge. Families of amino acid residues having side chainswith similar charges have been defined in the art. These familiesinclude amino acids with basic side chains (e.g., lysine, arginine,histidine), acidic side chains (e.g., aspartic acid, glutamic acid),uncharged polar side chains (e.g., glycine, asparagine, glutamine,serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g.,alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Alternatively, mutations can beintroduced randomly along all or part of the coding sequence, such as bysaturation mutagenesis, and the resultant mutants can be screened forbiological activity to identify mutants that retain activity (e.g., theability to bind an anti-factor XI polypeptide).

For example, it is possible to introduce mutations only in frameworkregions or only in CDR regions of an antibody molecule. Introducedmutations may be silent or neutral missense mutations, i.e., have no, orlittle, effect on an antibody's ability to bind antigen. These types ofmutations may be useful to optimize codon usage, or improve ahybridoma's antibody production. Alternatively, non-neutral missensemutations may alter an antibody's ability to bind antigen. The locationof most silent and neutral missense mutations is likely to be in theframework regions, while the location of most non-neutral missensemutations is likely to be in CDR, though this is not an absoluterequirement. One of skill in the art would be able to design and testmutant molecules with desired properties such as no alteration inantigen binding activity or alteration in binding activity (e.g.,improvements in antigen binding activity or change in antibodyspecificity). Following mutagenesis, the encoded protein may routinelybe expressed and the functional and/or biological activity of theencoded protein, (e.g., ability to immunospecifically bind at least oneepitope of a factor XI polypeptide) can be determined using techniquesdescribed herein or by routinely modifying techniques known in the art.

V. Fusion Proteins and Antibody Conjugates

Anti-factor XI monoclonal antibodies of the invention, orantigen-binding fragments, variants, or derivatives thereof, may furtherbe recombinantly fused to a heterologous polypeptide at the N- orC-terminus or chemically conjugated (including covalent and non-covalentconjugations) to polypeptides or other compositions. For example,anti-factor XI monoclonal antibodies may be recombinantly fused orconjugated to molecules useful as labels in detection assays andeffector molecules such as heterologous polypeptides, drugs,radionuclides, or toxins. See, e.g., PCT publications WO 92/08495; WO91/14438; WO 89/12624; U.S. Pat. No. 5,314,995; and EP 396,387.

The present invention also provides for fusion proteins comprising ananti-factor XI antibody, or antigen-binding fragment, variant, orderivative thereof, and a heterologous polypeptide. For example, theheterologous polypeptide to which the antibody is fused may be usefulfor function, may increase the in vivo half life of the polypeptides, ormay be a marker sequence that facilitates purification or detection(see, e.g., Leong et al. (2001) Cytokine 16:106; Adv. in Drug Deliv.Rev. (2002) 54:531; Weir et al. (2002) Biochem. Soc. Transactions30:512; Gentz et al. (1989) Proc. Natl. Acad. Sci. USA 86:821-824;Wilson et al. (1984) Cell 37:767)

Fusion proteins can be prepared using methods that are well known in theart (see for example U.S. Pat. Nos. 5,116,964 and 5,225,538). Theprecise site at which the fusion is made may be selected empirically tooptimize the binding characteristics of the fusion protein. DNA encodingthe fusion protein is then transfected into a host cell for expression.

Anti-factor XI monoclonal antibodies of the present invention, orantigen-binding fragments, variants, or derivatives thereof, may be usedin non-conjugated form or may be conjugated to at least one of a varietyof molecules, e.g., to improve the therapeutic properties of themolecule, to facilitate target detection, or for imaging or therapy of asubject. In particular, Anti-factor XI monoclonal antibodies of theinvention, or antigen-binding fragments, variants, or derivativesthereof, may be conjugated to therapeutic agents, prodrugs, peptides,proteins, enzymes, viruses, lipids, biological response modifiers,pharmaceutical agents, or PEG. Techniques for conjugating variousmoieties to an anti-factor XI antibody, or antigen-binding fragment,variant, or derivative thereof, are well known, see, e.g., Arnon et al.(1985) “Monoclonal Antibodies for Immunotargeting of Drugs in CancerTherapy,” in Monoclonal Antibodies and Cancer Therapy, ed. Reisfeld etal. (Alan R. Liss, Inc.), pp. 243-56; Hellstrom et al. (1987)“Antibodies for Drug Delivery,” in Controlled Drug Delivery, ed.Robinson et al. (2nd ed.; Marcel Dekker, Inc.), pp. 623-53); Thorpe(1985) “Antibody Carriers of Cytotoxic Agents in Cancer Therapy: AReview,” in Monoclonal Antibodies '84: Biological and ClinicalApplications, ed. Pinchera et al., pp. 475-506; “Analysis, Results, andFuture Prospective of the Therapeutic Use of Radiolabeled Antibody inCancer Therapy,” in Monoclonal Antibodies for Cancer Detection andTherapy, ed. Baldwin et al., Academic Press, pp. 303-16 (1985); andThorpe et al. (1982) “The Preparation and Cytotoxic Properties ofAntibody-Toxin Conjugates,” Immunol. Rev. 62:119-58.

VI. Polynucleotides Encoding Anti-Factor XI Antibodies

The present invention also provides for nucleic acid molecules encodinganti-factor XI antibodies of the invention, or antigen-bindingfragments, variants, or derivatives thereof.

In one embodiment, the present invention provides an isolatedpolynucleotide comprising, consisting essentially of, or consisting of anucleic acid encoding an immunoglobulin heavy chain domain (V_(H)domain), where at least one of the CDRs of the V_(H) domain has an aminoacid sequence that is at least about 80%, about 85%, about 90%, about95% , about 96%, about 97%, about 98%, about 99%, or identical to anyone of SEQ ID NOS:10-12.

In another embodiment, the present invention provides an isolatedpolynucleotide comprising, consisting essentially of, or consisting of anucleic acid encoding an immunoglobulin light chain domain (V_(L)domain), where at least one of the CDRs of the V_(L) domain has an aminoacid sequence that is at least about 80%, about 85%, about 90%, about95%, about 96%, about 97%, about 98%, about 99%, or identical to any oneof SEQ ID NOS:13-15.

In a further embodiment, the present invention includes an isolatedpolynucleotide comprising, consisting essentially of, or consisting of anucleic acid encoding a V_(H) domain that has an amino acid sequencethat is at least about 80%, about 85%, about 90%, about 91%, about 92%,about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about99%, or 100% identical to a reference V_(H) domain polypeptide sequenceselected from the group consisting of SEQ ID NO:3 and 7, wherein ananti-factor XI antibody comprising the encoded V_(H) domain specificallyor preferentially binds to factor XI.

In a further embodiment, the present invention includes an isolatedpolynucleotide comprising, consisting essentially of, or consisting of anucleic acid encoding a V_(L) domain that has an amino acid sequencethat is at least about 80%, about 85%, about 90%, about 91%, about 92%,about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about99%, or 100% identical to a reference V_(L) domain polypeptide sequenceselected from the group consisting of SEQ ID NO:5 and 9, wherein ananti-factor XI antibody comprising the encoded V_(L) domain specificallyor preferentially binds to factor XI.

In another embodiment, the present invention provides an isolatedpolynucleotide comprising, consisting essentially of, or consisting of anucleic acid that is at least about 80%, about 85%, about 90%, about91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%,about 98%, about 99%, or 100% identical to a reference polynucleotidesequence selected from the group consisting of SEQ ID NOS:2, 4, 6, and8.

Any of the polynucleotides described above may further includeadditional nucleic acids, encoding, e.g., a signal peptide to directsecretion of the encoded polypeptide, antibody constant regions asdescribed herein, or other heterologous polypeptides as describedherein. Also, as described in more detail elsewhere herein, the presentinvention includes compositions comprising one or more of thepolynucleotides described above. In one embodiment, the inventionincludes compositions comprising a first polynucleotide and secondpolynucleotide wherein said first polynucleotide encodes a V_(H) domainas described herein and wherein said second polynucleotide encodes aV_(L) domain as described herein. Specifically a composition whichcomprises, consists essentially of, or consists of a V_(H) domainselected from the group consisting of SEQ ID NO:3 and 7, and/or a V_(L)domain selected from the group consisting of SEQ ID NO:5 and 9.

The present invention also includes fragments of the polynucleotides ofthe invention, as described elsewhere. Additionally polynucleotides thatencode fusion polypolypeptides, Fab fragments, and other derivatives, asdescribed herein, are also contemplated by the invention.

The polynucleotides may be produced or manufactured by any method knownin the art. For example, if the nucleotide sequence of the antibody isknown, a polynucleotide encoding the antibody may be assembled fromchemically synthesized oligonucleotides (e.g., as described in Kutmeieret al. (1994) BioTechniques 17:242), which, briefly, involves thesynthesis of overlapping oligonucleotides containing portions of thesequence encoding the antibody, annealing and ligating of thoseoligonucleotides, and then amplification of the ligated oligonucleotidesby PCR.

Alternatively, a polynucleotide encoding an anti-factor XI antibody, orantigen-binding fragment, variant, or derivative thereof, may begenerated from nucleic acid from a suitable source. If a clonecontaining a nucleic acid encoding a particular antibody is notavailable, but the sequence of the antibody molecule is known, a nucleicacid encoding the antibody may be chemically synthesized or obtainedfrom a suitable source (e.g., an antibody cDNA library, or a cDNAlibrary generated front, or nucleic acid, preferably poly A+RNA,isolated front, any tissue or cells expressing the antibody or otheranti-factor XI antibody, such as hybridoma cells selected to express anantibody) by PCR amplification using synthetic primers hybridizable tothe 3′ and 5′ ends of the sequence or by cloning using anoligonucleotide probe specific for the particular gene sequence toidentify, e.g., a cDNA clone from a cDNA library that encodes theantibody or other anti-factor XI antibody. Amplified nucleic acidsgenerated by PCR may then be cloned into replicable cloning vectorsusing any method well known in the art.

Once the nucleotide sequence and corresponding amino acid sequence ofthe anti-factor XI antibody, or antigen-binding fragment, variant, orderivative thereof is determined, its nucleotide sequence may bemanipulated using methods well known in the art for the manipulation ofnucleotide sequences, e.g., recombinant DNA techniques, site directedmutagenesis, PCR, etc. (see, for example, the techniques described inSambrook et al. (1990) Molecular Cloning, A Laboratory Manual (2nd ed.;Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.) and Ausubel etal., eds. (1998) Current Protocols in Molecular Biology (John Wiley &Sons, NY), which are both incorporated by reference herein in theirentireties), to generate antibodies having a different amino acidsequence, for example to create amino acid substitutions, deletions,and/or insertions.

A polynucleotide encoding an anti-factor XI antibody, or antigen-bindingfragment, variant, or derivative thereof, can be composed of anypolyribonucleotide or polydeoxribonucleotide, which may be unmodifiedRNA or DNA or modified RNA or DNA. For example, a polynucleotideencoding anti-factor XI antibody, or antigen-binding fragment, variant,or derivative thereof can be composed of single- and double-strandedDNA, DNA that is a mixture of single- and double-stranded regions,single- and double-stranded RNA, and RNA that is mixture of single- anddouble-stranded regions, hybrid molecules comprising DNA and RNA thatmay be single-stranded or, more typically, double-stranded or a mixtureof single- and double-stranded regions. In addition, a polynucleotideencoding an anti-factor XI antibody, or antigen-binding fragment,variant, or derivative thereof can be composed of triple-strandedregions comprising RNA or DNA or both RNA and DNA. A polynucleotideencoding an anti-factor XI antibody, or antigen-binding fragment,variant, or derivative thereof, may also contain one or more modifiedbases or DNA or RNA backbones modified for stability or for otherreasons. “Modified” bases include, for example, tritylated bases andunusual bases such as inosine. A variety of modifications can be made toDNA and RNA; thus, “polynucleotide” embraces chemically, enzymatically,or metabolically modified forms.

An isolated polynucleotide encoding a non-natural variant of apolypeptide derived from an immunoglobulin (e.g., an immunoglobulinheavy chain portion or light chain portion) can be created byintroducing one or more nucleotide substitutions, additions or deletionsinto the nucleotide sequence of the immunoglobulin such that one or moreamino acid substitutions, additions or deletions are introduced into theencoded protein. Mutations may be introduced by standard techniques,such as site-directed mutagenesis and PCR-mediated mutagenesis.Preferably, conservative amino acid substitutions are made at one ormore non-essential amino acid residues.

VII. Expression of Antibody Polypeptides

DNA sequences that encode the light and the heavy chains of the antibodymay be made, either simultaneously or separately, using reversetranscriptase and DNA polymerase in accordance with well known methods.PCR may be initiated by consensus constant region primers or by morespecific primers based on the published heavy and light chain DNA andamino acid sequences. As discussed above, PCR also may be used toisolate DNA clones encoding the antibody light and heavy chains. In thiscase the libraries may be screened by consensus primers or largerhomologous probes, such as mouse constant region probes.

DNA, typically plasmid DNA, may be isolated from the cells usingtechniques known in the art, restriction mapped and sequenced inaccordance with standard, well known techniques set forth in detail,e.g., in the foregoing references relating to recombinant DNAtechniques. Of course, the DNA may be synthetic according to the presentinvention at any point during the isolation process or subsequentanalysis.

Following manipulation of the isolated genetic material to provideanti-factor XI antibodies, or antigen-binding fragments, variants, orderivatives thereof, of the invention, the polynucleotides encoding theanti-factor XI antibodies are typically inserted in an expression vectorfor introduction into host cells that may be used to produce the desiredquantity of anti-factor XI antibody.

Recombinant expression of an antibody, or fragment, derivative or analogthereof, e.g., a heavy or light chain of an antibody that binds to atarget molecule described herein, e.g., factor XI, requires constructionof an expression vector containing a polynucleotide that encodes theantibody. Once a polynucleotide encoding an antibody molecule or a heavyor light chain of an antibody, or portion thereof (preferably containingthe heavy or light chain variable domain), of the invention has beenobtained, the vector for the production of the antibody molecule may beproduced by recombinant DNA technology using techniques well known inthe art. Thus, methods for preparing a protein by expressing apolynucleotide containing an antibody encoding nucleotide sequence aredescribed herein. Methods that are well known to those skilled in theart can be used to construct expression vectors containing antibodycoding sequences and appropriate transcriptional and translationalcontrol signals. These methods include, for example, in vitrorecombinant DNA techniques, synthetic techniques, and in vivo geneticrecombination. The invention, thus, provides replicable vectorscomprising a nucleotide sequence encoding an antibody molecule of theinvention, or a heavy or light chain thereof, or a heavy or light chainvariable domain, operably linked to a promoter. Such vectors may includethe nucleotide sequence encoding the constant region of the antibodymolecule (see, e.g., PCT Publication WO 86/05807; PCT Publication WO89/01036; and U.S. Pat. No. 5,122,464) and the variable domain of theantibody may be cloned into such a vector for expression of the entireheavy or light chain.

The term “vector” or “expression vector” is used herein to mean vectorsused in accordance with the present invention as a vehicle forintroducing into and expressing a desired gene in a host cell. As knownto those skilled in the art, such vectors may easily be selected fromthe group consisting of plasmids, phages, viruses and retroviruses. Ingeneral, vectors compatible with the instant invention will comprise aselection marker, appropriate restriction sites to facilitate cloning ofthe desired gene and the ability to enter and/or replicate in eukaryoticor prokaryotic cells.

For the purposes of this invention, numerous expression vector systemsmay be employed. For example, one class of vector utilizes DNA elementsthat are derived from animal viruses such as bovine papilloma virus,polyoma virus, adenovirus, vaccinia virus, baculovirus, retroviruses(RSV, MMTV or MOMLV) or SV40 virus. Others involve the use ofpolycistronic systems with internal ribosome binding sites.Additionally, cells that have integrated the DNA into their chromosomesmay be selected by introducing one or more markers which allow selectionof transfected host cells. The marker may provide for prototrophy to anauxotrophic host, biocide resistance (e.g., antibiotics) or resistanceto heavy metals such as copper. The selectable marker gene can either bedirectly linked to the DNA sequences to be expressed, or introduced intothe same cell by cotransformation. Additional elements may also beneeded for optimal synthesis of mRNA. These elements may include signalsequences, splice signals, as well as transcriptional promoters,enhancers, and termination signals.

In particularly preferred embodiments the cloned variable region genesare inserted into an expression vector along with the heavy and lightchain constant region genes (preferably human) synthesized as discussedabove. Of course, any expression vector that is capable of elicitingexpression in eukaryotic cells may be used in the present invention.Examples of suitable vectors include, but are not limited to plasmidspcDNA3, pHCMV/Zeo, pCR3.1, pEF1/His, pIND/GS, pRc/HCMV2, pSV40/Zeo2,pTRACER-HCMV, pUB6/V5-His, pVAX1, and pZeoSV2 (available fromInvitrogen, San Diego, Calif.), and plasmid pCI (available from Promega,Madison, Wis.). In general, screening large numbers of transformed cellsfor those that express suitably high levels if immunoglobulin heavy andlight chains is routine experimentation that can be carried out, forexample, by robotic systems.

More generally, once the vector or DNA sequence encoding a monomericsubunit of the anti-factor XI antibody has been prepared, the expressionvector may be introduced into an appropriate host cell. Introduction ofthe plasmid into the host cell can be accomplished by various techniqueswell known to those of skill in the art. These include, but are notlimited to, transfection (including electrophoresis andelectroporation), protoplast fusion, calcium phosphate precipitation,cell fusion with enveloped DNA, microinjection, and infection withintact virus. See, Ridgway (1988) “Mammalian Expression Vectors” inVectors, ed. Rodriguez and Denhardt (Butterworths, Boston, Mass.),Chapter 24.2, pp. 470-472. Typically, plasmid introduction into the hostis via electroporation. The host cells harboring the expressionconstruct are grown under conditions appropriate to the production ofthe light chains and heavy chains, and assayed for heavy and/or lightchain protein synthesis. Exemplary assay techniques includeenzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), orfluorescence-activated cell sorter analysis (FACS), immunohistochemistryand the like.

The expression vector is transferred to a host cell by conventionaltechniques, and the transfected cells are then cultured by conventionaltechniques to produce an antibody for use in the methods describedherein. Thus, the invention includes host cells containing apolynucleotide encoding an antibody of the invention, or a heavy orlight chain thereof, operably linked to a heterologous promoter. Inpreferred embodiments for the expression of double-chained antibodies,vectors encoding both the heavy and light chains may be co-expressed inthe host cell for expression of the entire immunoglobulin molecule, asdetailed below.

As used herein, “host cells” refers to cells that harbor vectorsconstructed using recombinant DNA techniques and encoding at least oneheterologous gene. In descriptions of processes for isolation ofantibodies from recombinant hosts, the terms “cell” and “cell culture”are used interchangeably to denote the source of antibody unless it isclearly specified otherwise. In other words, recovery of polypeptidefrom the “cells” may mean either from spun down whole cells, or from thecell culture containing both the medium and the suspended cells.

A variety of host-expression vector systems may be utilized to expressantibody molecules for use in the methods described herein. Suchhost-expression systems represent vehicles by which the coding sequencesof interest may be produced and subsequently purified, but alsorepresent cells that may, when transformed or transfected with theappropriate nucleotide coding sequences, express an antibody molecule ofthe invention in situ. These include, but are not limited to,microorganisms such as bacteria (e.g., E. coli, B. subtilis) transformedwith recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expressionvectors containing antibody coding sequences; yeast (e.g.,Saccharomyces, Pichia) transformed with recombinant yeast expressionvectors containing antibody coding sequences; insect cell systemsinfected with recombinant virus expression vectors (e.g., baculovirus)containing antibody coding sequences; plant cell systems infected withrecombinant virus expression vectors (e.g., cauliflower mosaic virus,CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmidexpression vectors (e.g., Ti plasmid) containing antibody codingsequences; or mammalian cell systems (e.g., COS, CHO, BLK, 293, 3T3cells) harboring recombinant expression constructs containing promotersderived from the genome of mammalian cells (e.g., metallothioneinpromoter) or from mammalian viruses (e.g., the adenovirus late promoter;the vaccinia virus 7.5K promoter). Preferably, bacterial cells such asEscherichia coli, and more preferably, eukaryotic cells, especially forthe expression of whole recombinant antibody molecule, are used for theexpression of a recombinant antibody molecule. For example, mammaliancells such as Chinese hamster ovary cells (CHO), in conjunction with avector such as the major intermediate early gene promoter element fromhuman cytomegalovirus is an effective expression system for antibodies(Foecking et al. (1986) Gene 45:101; Cockett et al. (1990)Bio/Technology 8:2).

The host cell line used for protein expression is often of mammalianorigin; those skilled in the art are credited with ability topreferentially determine particular host cell lines that are best suitedfor the desired gene product to be expressed therein. Exemplary hostcell lines include, but are not limited to, CHO (Chinese Hamster Ovary),DG44 and DUXB11 (Chinese Hamster Ovary lines, DHFR minus), HELA (humancervical carcinoma), CVI (monkey kidney line), COS (a derivative of CVIwith SV40 T antigen), VERY, BHK (baby hamster kidney), MDCK, 293, WI38,R1610 (Chinese hamster fibroblast) BALBC/3T3 (mouse fibroblast), HAK(hamster kidney line), SP2/O (mouse myeloma), P3x63-Ag3.653 (mousemyeloma), BFA-1c1BPT (bovine endothelial cells), RAJI (human lymphocyte)and 293 (human kidney). Host cell lines are typically available fromcommercial services, the American Tissue Culture Collection or frompublished literature.

In addition, a host cell strain may be chosen that modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells that possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines that stably express theantibody molecule may be engineered. Rather than using expressionvectors that contain viral origins of replication, host cells can betransformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which stably express theantibody molecule.

A number of selection systems may be used, including, but not limitedto, the herpes simplex virus thymidine kinase (Wigler et al. (1977) Cell11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska andSzybalski (1992) Proc. Natl. Acad. Sci. USA 48:202), and adeninephosphoribosyltransferase (Lowy et al. (1980) Cell 22:817) genes can beemployed in tk-, hgprt- or aprt-cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al. (1980) Natl. Acad. Sci. USA 77:357; O'Hare et al. (1981) Proc.Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance tomycophenolic acid (Mulligan and Berg (1981) Proc. Natl. Acad. Sci. USA78:2072); neo, which confers resistance to the aminoglycoside G-418Clinical Pharmacy 12:488-505; Wu and Wu (1991) Biotherapy 3:87-95;Tolstoshev (1993) Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan(1993) Science 260:926-932; and Morgan and Anderson (1993) Ann. Rev.Biochem. 62:191-217 (1993); TIB TECH 11(5):155-215 (May, 1993); andhygro, which confers resistance to hygromycin (Santerre et al. (1984)Gene 30:147. Methods commonly known in the art of recombinant DNAtechnology which can be used are described in Ausubel et al. (1993)Current Protocols in Molecular Biology (John Wiley & Sons, NY); Kriegler(1990) “Gene Transfer and Expression” in A Laboratory Manual (StocktonPress, NY); Dracopoli et al. (eds) (1994) Current Protocols in HumanGenetics (John Wiley & Sons, NY) Chapters 12 and 13; Colberre-Garapin etal. (1981) J. Mol. Biol. 150:1, which are incorporated by referenceherein in their entireties.

The expression levels of an antibody molecule can be increased by vectoramplification (for a review, see Bebbington and Hentschel (1987) “TheUse of Vectors Based on Gene Amplification for the Expression of ClonedGenes in Mammalian Cells in DNA Cloning” (Academic Press, NY) Vol. 3.When a marker in the vector system expressing antibody is amplifiable,increase in the level of inhibitor present in culture of host cell willincrease the number of copies of the marker gene. Since the amplifiedregion is associated with the antibody gene, production of the antibodywill also increase (Crouse et al. (1983) Mol. Cell. Biol. 3:257).

In vitro production allows scale-up to give large amounts of the desiredpolypeptides. Techniques for mammalian cell cultivation under tissueculture conditions are known in the art and include homogeneoussuspension culture, e.g. in an airlift reactor or in a continuousstirrer reactor, or immobilized or entrapped cell culture, e.g. inhollow fibers, microcapsules, on agarose microbeads or ceramiccartridges. If necessary and/or desired, the solutions of polypeptidescan be purified by the customary chromatography methods, for example gelfiltration, ion-exchange chromatography, chromatography overDEAE-cellulose or (immuno-)affinity chromatography, e.g., afterpreferential biosynthesis of a synthetic hinge region polypeptide orprior to or subsequent to the HIC chromatography step described herein.

Genes encoding anti-factor XI antibodies, or antigen-binding fragments,variants, or derivatives thereof of the invention can also be expressedin non-mammalian cells such as bacteria or yeast or plant cells.Bacteria that readily take up nucleic acids include members of theenterobacteriaceae, such as strains of Escherichia coli or Salmonella;Bacillaceae, such as Bacillus subtilis; Pneumococcus; Streptococcus, andHaemophilus influenzae. It will further be appreciated that, whenexpressed in bacteria, the heterologous polypeptides typically becomepart of inclusion bodies. The heterologous polypeptides must beisolated, purified and then assembled into functional molecules. Wheretetravalent forms of antibodies are desired, the subunits will thenself-assemble into tetravalent antibodies (WO 02/096948A2).

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the antibodymolecule being expressed. For example, when a large quantity of such aprotein is to be produced, for the generation of pharmaceuticalcompositions of an antibody molecule, vectors which direct theexpression of high levels of fusion protein products that are readilypurified may be desirable. Such vectors include, but are not limited, tothe E. coli expression vector pUR278 (Ruther et al. (1983) EMBO J.2:1791), in which the antibody coding sequence may be ligatedindividually into the vector in frame with the lacZ coding region sothat a fusion protein is produced; pIN vectors (Inouye and Inouye (1985)Nucleic Acids Res. 13:3101-3109; Van Heeke and Schuster (1989) J. Biol.Chem. 24:5503-5509); and the like. pGEX vectors may also be used toexpress foreign polypeptides as fusion proteins with glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption and binding to amatrix glutathione-agarose beads followed by elution in the presence offree glutathione. The pGEX vectors are designed to include thrombin orfactor Xa protease cleavage sites so that the cloned target gene productcan be released from the GST moiety.

In addition to prokaryotes, eukaryotic microbes may also be used.Saccharomyces cerevisiae, or common baker's yeast, is the most commonlyused among eukaryotic microorganisms although a number of other strainsare commonly available, e.g., Pichia pastoris.

For expression in Saccharomyces, the plasmid YRp7, for example,(Stinchcomb et al. (1979) Nature 282:39; Kingsman et al. (1979) Gene7:141; Tschemper et al. (1980) Gene 10:157) is commonly used. Thisplasmid already contains the TRP1 gene, which provides a selectionmarker for a mutant strain of yeast lacking the ability to grow intryptophan, for example ATCC No. 44076 or PEP4-1 (Jones (1977) Genetics85:12). The presence of the trp1 lesion as a characteristic of the yeasthost cell genome then provides an effective environment for detectingtransformation by growth in the absence of tryptophan.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is typically used as a vector to express foreign genes. Thevirus grows in Spodoptera frugiperda cells. The antibody coding sequencemay be cloned individually into non-essential regions (for example thepolyhedrin gene) of the virus and placed under control of an AcNPVpromoter (for example the polyhedrin promoter).

Once an antibody molecule of the invention has been recombinantlyexpressed, it may be purified by any method known in the art forpurification of an immunoglobulin molecule, for example, bychromatography (e.g., ion exchange, affinity, particularly by affinityfor the specific antigen after Protein A, and sizing columnchromatography), centrifugation, differential solubility, or by anyother standard technique for the purification of proteins.Alternatively, a preferred method for increasing the affinity ofantibodies of the invention is disclosed in U.S. Patent ApplicationPublication No. 2002 0123057 A1.

VIII. Methods of Use for Anti-Factor XI Monoclonal Antibodies

Methods of the invention are directed to the use of anti-factor XImonoclonal antibodies, including antigen-binding fragments, variants,and derivatives thereof, to inhibit thrombosis in a subject in needthereof. Though the following discussion refers to methods and treatmentof various diseases and disorders with anti-factor XI monoclonalantibody of the invention, the methods described herein are alsoapplicable to the antigen-binding fragments, variants, and derivativesof these anti-factor XI monoclonal antibodies that retain the desiredproperties of the anti-factor XI monoclonal antibodies of the invention,i.e., capable of specifically binding human factor XI and being able toinhibit the coagulation activity of human factor XI.

Anti-factor XI monoclonal antibodies of the invention may beadministered to any subject in which inhibition of thrombosis would bebeneficial. In particular, it is contemplated that the anti-factor XImonoclonal antibodies of the invention are particularly useful asantithrombotic agents that do not compromise hemostasis, and as such aresuperior alternatives to traditional antithrombotic agents.

In one embodiment, the anti-factor XI monoclonal antibodies of theinvention may be used to treat conditions characterized by vascularocclusions, such as those that occur as a result of thrombus formation.Conditions that are characterized by vascular occlusions and justifytreatment or prevention using anti-factor XI monoclonal antibodies ofthe include those that involve the arterial, capillary, and venousvasculature.

In the coronary arteries, occlusive thrombus formation often follows therupture of atherosclerotic plaque. This occlusion is the major cause ofacute myocardial infarction and unstable angina. Coronary occlusions canalso occur following infections, inflammation, thrombolytic therapy,angioplasty, and graft placements. Similar principles apply to otherparts of the arterialvasculature and include, among others, thrombusformation in the carotid arteries, which is the major cause of transientor permanent cerebral ischemia and stroke.

Venous thrombosis often follows stasis, infections, inflammatoryreactions, and major surgery of the lower extremities or the abdominalarea. Deep vein thrombosis results in reduced blood flow from the areadistal to the thrombus and predisposes to pulmonary embolism. Pulmonaryembolism is a major cause of post-surgical mortality. Disseminatedintravascular coagulation (DIC) and acute respiratory distress syndrome(ARDS) where the monoclonal antibodies of this invention are usefulcommonly occur within all vascular systems during bacterial sepsis,entry of foreign material into the blood stream following, e.g., traumaand child birth, immune reactions, inflammation, certain viralinfections, certain platelet disorders, and cancer. Disseminatedintravascular coagulation is a severe complication of many diseaseconditions and some drug treatments, including, for example, hepatin.Thrombotic consumption of coagulation factors and platelets, andsystemic coagulation results in the formation of life-threateningthrombi occurring throughout the microvasculature leading to local orwidespread hypoxia and organ failure.

Thus, in one embodiment, a method is provided for inhibiting thrombosisin a subject in need thereof comprising administering to the subject aneffective dose of an anti-factor XI monoclonal antibody of theinvention, particularly where the thrombosis is associated with: 1)acute coronary syndromes such as myocardial infarction, unstable angina,refractory angina, occlusive coronary thrombus occurringpost-thrombolytic therapy or post-coronary angioplasty; 2) ischemiccerebrovascular syndromes including embolic stroke, thrombotic stroke,or transient ischemic attacks; 3) thrombosis occurring in the venoussystem occurring either spontaneously or in the setting of malignancy,trauma, or surgery, including pulmonary thromboembolism; 4) anycoagulopathy including ARDS and DIC, e.g., in the setting of sepsis orother infection, surgery, pregnancy, trauma, or malignancy and whetherassociated with multi-organ failure or not, thrombotic thrombocytopenicpurpura, thromboangiitis obliterans, or thrombotic disease associatedwith heparin induced thrombocytopenia; 5) thrombotic complicationsassociated with extracorporeal circulation (e.g., renal dialysis,cardiopulmonary bypass or other oxygenation procedure, andplasmaphoresis); 6) thrombotic complications associated withinstrumentation (e.g. cardiac or other intravascular catheterization,intraaortic balloon pump, and coronary stent or cardiac valve); and 7)complications associated with fitting of prosthetic devices.

As described elsewhere herein, traditional antithrombotic agents aredangerous or even fatal when administered at their maximally effectivedoses. Accordingly, in another embodiment, a method is provided forreducing a required dose or complementing the effect of anantithrombotic agent in the treatment of thrombosis in a subject in needthereof comprising administering to the subject an effective dose of ananti-factor XI monoclonal antibody of the invention. Traditionalantithrombotic agents include direct or indirect thrombin inhibitor, aFactor X inhibitor, a Factor IX inhibitor, a Factor XII inhibitor, aFactor V inhibitor, a Factor VIII inhibitor, a Factor XIII inhibitor, aFactor VII inhibitor, a tissue factor inhibitor, a profibrinolyticagent, a fibrinolytic or fibrinogenolytic agent, a carboxypeptidase Binhibitor, a platelet inhibitor, a selective platelet count reducingagent, or a Factor XI inhibitor.

Direct thrombin inhibitors include argatroban and derivatives or analogsthereof, hirudin and recombinant or synthetic derivatives or analogsthereof, derivatives of the tripeptide Phe-Pro-Arg, chloromethylketonederivatives, ximelagatran and derivatives, metabolites, or analogsthereof, anion binding exosite inhibitors, and RNA/DNA aptamers.

Indirect thrombin inhibitors include heparin, coumarin, dermatan, andthrombomodulin.

Factor X inhibitors include direct factor Xa inhibitors, rivaroxaban,antibodies to factor X, inactivated factor Xa, or analogs andderivatives thereof.

Factor IX inhibitors include antibodies to factor IX, direct factor IXainhibitors, or inactivated factor IXa, or analogs and derivativesthereof.

Factor XII inhibitors include direct factor XII inhibitors, corn trypsininhibitor, antibodies to FXII, or inactivated factor XIIa or analogs andderivatives thereof.

Factor V inhibitors include antibodies to factor V, activated protein C,protein S, or analogs and derivatives thereof.

Factor VIII inhibitors include antibodies to FVIII, activated protein C,protein S, or analogs and derivatives thereof.

Factor XIII inhibitors include antibodies to factor XIII, direct factorXIIIa inhibitors, or inactivated factor XIIIa.

Factor VII inhibitors include antibodies to factor VII, tissue factorpathway inhibitor, inactivated factor VIIa, or direct factor VIIainhibitor or analogs and derivatives thereof.

Tissue factor inhibitors include tissue factor pathway inhibitor,antibodies to tissue factor, or analogs and derivatives thereof.

Profibrinolytic agents include urokinase, streptokinase, tissueplasminogen activator or derivatives thereof.

Fibrinolytic or fibrinogenolytic agents include plasmin, microplasmin,ancrod, or derivatives thereof.

Platelet inhibitors include aspirin, clopidogrel, dypiridamol, orderivatives thereof.

Selective platelet count reducing agents include hydroxyurea,anagrelide, or derivatives thereof.

Factor XI inhibitors include direct factor XIa inhibitors, otherantibodies to factor XI, inactivated factor XIa, or analogs andderivatives thereof.

In another embodiment, a method is provided for treating metastaticcancer in a subject in need thereof comprising administering to thesubject an effective dose of an anti-factor XI monoclonal antibody ofthe invention.

In yet another embodiment, a method is provided for treating an acuteinflammatory reaction in a subject in need thereof comprisingadministering to the subject an effective dose of an anti-factor XImonoclonal antibody of the invention.

In further embodiments of the present invention, combination therapiesare provided in which an anti-factor XI monoclonal antibody is theprimary active agent and is administered along with an additional activeagent to a subject in need thereof. Such combination therapy may becarried out by administration of the different active agents in a singlecomposition, by concurrent administration of the different active agentsin different compositions, or by sequential administration of thedifferent active agents. The combination therapy may also includesituations where the anti-factor XI monoclonal antibody is already beingadministered to the patient, and the additional active agent is to beadded to the patient's drug regimen, as well as where differentindividuals (e.g., physicians or other medical professionals) areadministering the separate components of the combination to the patient.

The additional active agent will generally, although not necessarily, beone that is effective in inhibiting thrombosis. In a preferredembodiment, the additional active agent is a hemostatic agent, i.e., anagent that promotes hemostasis. Particularly preferred hemostatic agentsfor use in the combination therapies of the present invention includeactivated factor VII (FVIIa) or activated prothrombin complexconcentrate (APCC). Both FVIIa and APCC were developed as hemostaticagents for the treatment of bleeding in patients withinhibitor-developing hemophilia (Scharrer (1999) Haemophilia, 5:253-259;Shapiro et al. (1998) Thromb. Haemost., 80:773-778; Lusher et al. (1980)N. Engl. J. Med., 303:421-425; Sjamsoedin et al. (1981) N. Engl. J.Med., 305:717-21; Negrier et al. (1997) Thromb. Haemost., 77:1113-1119).The key active ingredient of APCC is prothrombin, which contributes toboth hemostasis and thrombus growth (Akhavan et al. (2000) Thromb.Haemost., 84:989-997; Xi et al. (1989) Thromb. Haemost., 62:788-791). Bycontrast, increasing the plasma concentration of FVIIa is thought toincrease the generation of thrombin predominantly through a tissuefactor (TF) dependent pathway in which the TF/FVIIa complex activatesfactors IX and X (Hoffman and Monroe (2001) Thromb. Haemost.,85:958-965).

IX. Pharmaceutical Compositions and Administration Methods

The anti-factor XI monoclonal antibodies of the invention, orantigen-binding fragments, variants, or derivatives thereof, can beformulated according to known methods for preparing pharmaceuticallyuseful compositions, such as by admixture with a pharmaceuticallyacceptable carrier vehicle. Suitable vehicles and their formulations aredescribed, for example, in Remington's Pharmaceutical Sciences (16thed., Osol, A. (ed.), Mack, Easton Pa. (1980)). In order to form apharmaceutically acceptable composition suitable for effectiveadministration, such compositions will contain an effective amount ofthe anti-factor XI monoclonal antibody, or antigen-binding fragments,variants, or derivatives thereof, either alone, or with a suitableamount of carrier vehicle.

The term “pharmaceutically acceptable” as used herein refers to atherapeutic agent or compound that while biologically active will notdamage the physiology of the recipient human or animal to the extentthat the viability of the recipient is comprised.

Pharmaceutical compositions may be administered in admixture with asuitable carrier, diluent, or excipient such as sterile water,physiological saline, glucose, or the like. The compositions can containauxiliary substances such as wetting or emulsifying agents, pH bufferingagents, adjuvants, gelling or viscosity enhancing additives,preservatives, flavoring agents, colors, and the like, depending uponthe route of administration and the preparation desired.

Pharmaceutical compositions may be formulated for immediate release orcontrolled release. The “absorption pool” represents a solution of thedrug administered at a particular absorption site, and k_(r), k_(a), andk_(e) are first-order rate constants for: 1) release of the drug fromthe formulation; 2) absorption; and 3) elimination, respectively. Forimmediate release dosage forms, the rate constant for drug release k_(r)is far greater than the absorption rate constant k_(a). For controlledrelease formulations, the opposite is true, i.e., k_(r)<<<k_(a), suchthat the rate of release of drug from the dosage form is therate-limiting step in the delivery of the drug to the target area. Theterm “controlled release” as used herein includes any nonimmediaterelease formulation, including but not limited to sustained release,delayed release and pulsatile release formulations.

Pharmaceutical compositions comprising anti-factor XI monoclonalantibodies of the invention, or antigen-binding fragments, variants, orderivatives thereof, can be administered in dosages and by techniqueswell known to those skilled in the medical or veterinary arts, takinginto consideration such factors as the age, sex, weight, species andcondition of the particular subject, and the route of administration.The route of administration can be via any route that delivers a safeand therapeutically effective dose of an anti-factor XI monoclonalantibody of the invention, or antigen-binding fragments, variants, orderivatives thereof, to the blood of an animal or human. Forms ofadministration, include, but are not limited to, systemic, topical,enteral, and parenteral routes of administration. Enteral routes includeoral and gastrointestinal administration. Parenteral routes includeintravenous, intraarterial, intramuscular, intraperitoneal,subcutaneous, transdermal, and transmucosal administration. Other routesof administration include epidural or intrathecal administration.

The effective dosage and route of administration are determined by thetherapeutic range and nature of the compound, and by known factors, suchas the age, weight, and condition of the subject, as well as LD₅₀ andother screening procedures that are known and do not require undueexperimentation.

The term “dosage” as used herein refers to the amount of an anti-factorXI monoclonal antibody of the invention, or antigen-binding fragments,variants, or derivatives thereof, administered to an animal or human.The therapeutic agent may be delivered to the recipient as a bolus or bya sustained (continuous or intermittent) delivery. When the delivery ofa dosage is sustained over a period, which may be in the order of a fewminutes to several days, weeks or months, or may be administerchronically for a period of years, the dosage may be expressed as weightof the therapeutic agent/kg body weight of the patient/unit time ofdelivery.

By “therapeutically effective dose or amount” or “effective amount” isintended an amount of an anti-factor XI monoclonal antibody that whenadministered brings about a positive therapeutic response with respectto treatment of a patient in need thereof. In some embodiments of theinvention, a therapeutically effective dose of the anti-factor XImonoclonal antibody is in the range from about 0.01 mg/kg to about 10mg/kg, from about 0.1 mg/kg to about 10 mg/kg, from about 1 mg/kg toabout 10 mg/kg, from about 1 mg/kg to about 5 mg/kg, from about 0.01mg/kg to about 1 mg/kg, or from about 0.1 mg/kg to about 1 mg/kg. Inother embodiments, the therapeutically effective doses of anti-factor XImonoclonal antibody, is about 0.01 mg/kg, about 0.03 mg/kg, about 0.1mg/kg, about 0.3 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 1.5 mg/kg,about 2 mg/kg, about 2.5 mg/kg, about 3 mg/kg, about 5 mg/kg, about 7mg/kg, about 10 mg/kg, or other such doses falling within the range ofabout 0.01 mg/kg to about 10 mg/kg. It is recognized that the method oftreatment may comprise a single administration of a therapeuticallyeffective dose or multiple administrations of a therapeuticallyeffective dose of the anti-factor XI monoclonal antibody.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL Example 1. Inhibition of Factor XI with Monoclonal Antibody1A6 Decreases Thrombin Production and Prevents Vascular Occlusion inExperimental Thrombosis in Primates

The present study in primates was designed to help clarify the role ofFXI in thrombus formation, and to determine if FXI inhibition representsa relatively safe thrombosis specific approach for limiting thrombuspropagation. By measuring local procoagulant and fibrinolytic markers,uniquely sensitive time dependent measurements of local thrombinproduction, platelet activation, and fibrinolysis were assessed. Inorder to delineate the role of FXI, studies were also performed using apotent monospecific antibody to FXI. Platelet and fibrin deposition onthrombogenic devices were assessed, and vascular graft occlusion studieswere performed to determine the importance of FXI in the propagation andstability of relatively large experimental thrombi under arterial typeshear. These results demonstrate for the first time in vivo that FXI iscapable of facilitating robust thrombin generation and plateletactivation on the flow surface of forming thrombi, which contributesdirectly to thrombus growth and stability. These results also show thatFXI related inhibition of fibrinolysis plays, at most, a minor role inlimiting the degradation of rapidly propagating arterial type thrombi.

Methods

Experimental Animals.

A total of 39 non-terminal studies were performed on 17 normal juvenilemale baboons (Papio anubis) weighing 9-11 kg. Experiments were conductedon non-anticoagulated awake animals that had chronic exteriorizedarterio-venous (AV) shunts previously placed between the femoral arteryand vein, as described elsewhere (Hanson et al. (1993) J. Clin. Invest.92:2003-2012). Baseline shunt blood flow exceeded 250 ml/min in allstudy animals. Anxiety was managed with low dose ketamine (<2 mg/kg/hr).Platelet counts, red cell counts, and hematocrits were measured daily,before and after the experiments. Calculated blood loss did not exceed4% of total blood volume on any experimental day.

Thrombosis Model.

Thrombus formation was initiated within the baboon AV shunt byinterposing a thrombogenic segment of prosthetic vascular graft (ePTFE,WL Gore & Co., Flagstaff, Ariz.), as previously described (Hanson et al.(1993) J. Clin. Invest. 92:2003-2012). To consistently triggerplatelet-dependent thrombus formation, the clinical graft segments werecoated with immobilized collagen. Twenty mm long grafts having internaldiameters (i.d.) of either 2 or 4 mm were filled with equine type Icollagen (1 mg/ml; Nycomed Arzenmittel, Munich, Germany) for 15 min, andthen dried overnight under sterile airflow. This method produced auniform collagen coating within the graft lumen as determined byscanning electron microscopy (FIGS. 1B and 1C). The thrombogeniccollagen-coated grafts were then incorporated between segments ofsilicon rubber tubing, and deployed into the AV shunts (FIG. 1A). Thegrafts were exposed to blood for up to 60 min. During each experiment,the blood flow rate through the graft was restricted to 100 ml/min byclamping the proximal silicone rubber shunt segment, thereby producing amean wall shear rate (MWSR) in the 4 mm grafts of 265 s⁻¹, while in the2 mm grafts the initial MWSR was 2120 s⁻¹. Flow rates were continuouslymonitored using an ultrasonic flow meter (Transonics Systems, Ithaca,N.Y.). The 4 mm grafts did not occlude and pulsatile flow rates remainedat 100 ml/min until the thrombogenic graft segments were removed at 60min. Baseline blood flow was restored through the permanent shunt aftereach experiment. In the 2 mm diameter grafts blood flow ratesprogressively declined due to thrombus formation. The grafts wereremoved from the AV shunts when the flow rate fell from 100 ml/min tobelow 20 ml/min, signaling imminent occlusion. The time from initiationof blood flow to graft removal (<20 ml/min blood flow) was taken as theocclusion time.

For imaging of the platelet deposition, autologous baboon platelets werelabeled with 1 mCi of ¹¹¹In-oxine as previously described (Hanson et al.(1993) J. Clin. Invest. 92:2003-2012). Labeled platelets were infusedand allowed to circulate for at least 1 h before studies were performed.Accumulation of labeled platelets onto thrombogenic grafts were measuredin 5-min intervals using a gamma scintillation camera. Homologous¹²⁵I-labeled baboon fibrinogen (4 μCi, >90% clottable) was infused 10min before each study, and incorporation of the labeled fibrin withinthe thrombus was assessed using a gamma counter 30 days later to allowthe ¹¹¹In to decay. The radioactivity deposited (cpm) was divided by theclottable fibrin(ogen) radioactivity of samples taken at the time of theoriginal study (cpm/mg).

Occlusion studies were performed using 10 mm long 2 mm i.d. collagencoated devices which produced high arterial shear rates (2120 s⁻¹ at 100ml/min clamped blood flow). Accumulation of labeled platelets onto 2 mmthrombogenic grafts were measured in 3-min intervals using a gammascintillation camera. Flow was maintained at 100 ml/min by proximalclamping for as long as possible, and then allowed to decrease as thepropagating thrombus began to occlude the device. A final blood flowrate of 20 ml/min was used as a cutoff for occlusion, since a fullyocclusive thrombi and lack of blood flow through the device could leadto occlusion of the shunt and a significant loss of blood for theanimals.

Sampling Method.

A novel local sampling method was used to assess the local production ofthrombogenic mediators by sampling blood that superfused propagatingexperimental thrombi. Since thrombotic markers can be rapidly degradedand cleared from circulation, systemic sampling may lack the sensitivityto detect more subtle changes, and does not establish the location ofsuch marker generation. All blood samples were collected into 1/10^(th)volume 3.8% citrate anticoagulant. Systemic samples (1 ml each) werecollected before initiation of thrombosis as well as 30 and 60 min intoeach study. A total of 6 local blood samples were drawn from theperipheral blood IBL at a rate of 100 μl/min over 10 min intervals outof a 0.64 mm port which was located 10 mm distal to the growing thrombus(FIG. 1A). In order to maintain patency of the sample port,Phe-Pro-Arg-chloromethylketone (PPACK, 0.5 mg/ml) anticoagulant, whichdirectly inhibits thrombin, FVIIa, FXa, FXIa, and various other serineproteases (Angliker et al. (1988) Biochem J. 256:481-486), was infusedimmediately upstream at a rate of 20 μl/min, also using a 0.64 mm port.Infusion and sampling were regulated by syringe pumps (HarvardApparatus). Local sampling was performed only with the 4 mm i.d. devicesin this study. PPACK did not affect systemic prothrombin times (PT) oractivated partial thromboplastin times (aPTT) that were measured insequential plasma samples, nor did local sampling with PPACK affectplatelet or fibrin deposition onto collagen-coated grafts as comparedwith historical results in this model.

For control studies, normal length silicone rubber extension tubing wasused without inserting a thrombogenic device. The tubing was cut andreattached in the same location of graft insertion. Sample andanticoagulant infusion ports were positioned as described above, andblood samples were collected using the same approach. This provided acontrol of the local sampling technique to determine if this methodproduced activation of platelets and the coagulation pathway independentof the thrombogenic device. Consistent with previous studies (Savage etal. (1986) Blood 68:386-393), the silicone tubing was not measurablythrombogenic; neither the silicone polymer nor the sampling techniqueemployed produced significant coagulation pathway or plateletactivation.

Blood Sample Analysis.

Blood cell counts were determined using a micro-60 automated cellcounter (Horiba-ABX Diagnostics). Blood samples were divided into twoaliquots and processed according to specific test requirements. Allsamples were placed on ice for 10 min, centrifuged at 4° C. for 10 minat 12,900 g, and the plasmas were stored at −80° C. Forβ-thromboglobulin (βTG) determinations, the samples were supplementedwith 4 μg/ml prostaglandin E1 (PGE₁), 4.3 mg/ml acetylsalicylic acid(ASA), and 50 μg/ml PPACK. Cross-reacting ELISA assays were used todetermine D-dimer levels (IMUCLONE® D-Dimer, American Diagnostica; LOD:35 ng/mL), the platelet activation marker βTG (Asserachrom®, DiagnosticaStago; LOD: 5 IU/mL), and thrombin-antithrombin complexes (TAT,Enzygnost-TAT, Dade-Behring; LOD: 2 ng/mL). All ELISA test kits utilizedfor these studies have previously shown sensitivity to baboon markers.

Factor XI and Platelet Inhibition In Vivo.

Since previous studies showed that polyclonal antibodies to human FXIrequired very high doses to achieve inhibition of FXI in baboons (Gruberet al. (2003) Blood 102:953-955), a new reagent was generated: a potentneutralizing anti-human FXI monoclonal antibody (aXIMab) thatcross-reacted with baboon FXI. Hybridomas were derived from Balb/c miceimmunized with purified human FXI using standard procedures (de StGroth& Scheidegger (1980) J. Immunol. Methods 35:1-21). Hybridomas werescreened using solid phase ELISA against human FXI, and those thatshowed binding were subcloned twice by limiting dilution. The clone thatproduced the most potent neutralizing antibody, which inhibited both theactivation of FXI and the activity of FXIa, was selected based onprolongation of the clotting time of recalcified normal human plasma(NHP) and normal baboon plasma (NBP) by the cell culture supernatant.The cell line producing aXIMab (1A6) was grown in a CL1000 bioreactoraccording to the manufacturer's protocol (Integra Biosciences), and theantibody was purified from the media using cation exchange and protein Achromatography.

Human and baboon FXI in plasma was recognized by aXIMab as a single bandat 160 kDa on Western blots (FIG. 7A). The antibody specificallyrecognized the third apple (A3) domain of the FXI heavy chain, asassessed by immunoblotting of recombinant FXI/prekallikrein chimeras(see Example 2 below; see also Sun et al. (1996) J. Biol. Chem.271:29,023-29,028). The IC₅₀ and IC₉₉ of aXIMab in vitro was 2.5 nM and10 nM, respectively, in a clotting assay using FXI deficient humanplasma (George King Bio-Medical) with serial dilutions of NBP asstandards (Proctor et al. (1961) Am. J. Clin. Pathol. 36:212-219).Purified aXIMab (monoclonal antibody 1A6), tested within a concentrationrange from 0-40 nM, prolonged the activated partial thromboplastin time(aPTT) (Hemosil™ SynthASil, Instrumentation Laboratory) similarly inboth NHP and NBP in a concentration-dependent manner without affectingthe prothrombin time (PT) (Innovin®, Dade Behring).

Pharmacological Inhibition of FXI and Platelet Activities In Vivo.

In a pilot experiment, aXIMab (monoclonal antibody 1A6; 2 mg/kg) wasadministered to a single baboon, and blood samples were collected intocitrate anticoagulant over 4 weeks to measure circulating FXI antigen(FXI:Ag) concentrations, FXI inhibitor, and FXI procoagulant activity ofeach sample. This dose of aXIMab was chosen with the intent to achievesustained and near-complete inhibition of FXI, assuming an initialdilution of the antibody into 60 ml blood volume per kg body weightafter injection. The maximum achievable prolongation of the aPTT wasabout 2.5 fold. FXI:Ag was measured by ELISA using goat anti-human FXIpolyclonal capture and detection (horseradish peroxidase conjugated[HRP]) antibodies (Affinity Biologicals, Hamilton, Ontario), which alsorecognized baboon FXI and its complex with aXIMab. A standard curve wasconstructed with serial dilutions of NBP, and FXI concentrations weredetermined in percentage of NBP. Western blots for FXI were performed bysize fractionation of 1 μl samples of plasma under non-reducingconditions on 7.5% polyacrylamide-SDS gels, followed by transfer to PVDFmembranes. Detection was with a goat-anti-human FXI polyclonal antibodyconjugated to HRP and chemiluminescence. In the same samples, theBethesda assay (Kasper et al. (1975) Thromb. Diath. Haemorrh.34:869-872) was used to determine excess (non-complexed) circulating FXIinhibitor (aXIMab) activity levels, and the FXI procoagulant activitywas assayed using a clotting assay (Proctor et al. (1961) Am. J. Clin.Pathol. 36:212-219).

aXIMab was administered as a single bolus (2 mg/kg intravenously) atleast 24 h before the thrombosis experiments. The anticoagulant effect(aPTT) was monitored daily, and thrombosis experiments were performedwhile the FXI procoagulant activity in the circulating blood was reducedby ≥99%, as assessed by comparing the clotting times to a standard curvegenerated with FXI deficient human plasma (George King Bio-Medical). ThePT was also assessed in all baboons and no differences were seen betweengroups.

We previously showed that inhibition of FXI by polyclonal antibodies issafer and as effective as high dose heparin in baboons (Gruber & Hanson(2003) Blood 102:953-955). Since aspirin is a less antihemostatic agentthan heparin, and it is often included in the standard treatment ofarterial-type platelet-dependent thrombosis, ASA was used in the currentstudies with the occlusion-prone 2 mm i.d. grafts as a long-actingpositive control. ASA (32 mg/kg) was administered orally 2-4 h beforeeach thrombosis experiment, as described previously (Hanson et al.(1985) J. Clin. Invest. 75:1591-1599). Four weeks were allowed forwashout of each aXIMab and ASA before performing new experiments in thesame animal.

Hemostatic Assessment.

The effects of FXI inhibition and aspirin on primary hemostasis inbaboons were assessed using the standard template skin bleeding timetest (Surgicutt®, International Technidyne Corp). Experimentally, thisand similar tests (e.g., Simplate bleeding times) have been shown to besensitive to the effects of therapeutic anticoagulants, anti-plateletagents, and coagulation abnormalities in humans and non-human primates(Gruber et al. (2007) Blood 109:3733-3740; Smith et al. (1985) Am. J.Clin. Pathol. 83:211-215; Payne et al. (2002) J. Vasc. Surg.35:1204-1209). All bleeding time measurements were performed by the sameexpert technician. For indirect assessment of hemostasis, aPTT and PTmeasurements were also performed.

Computational Modeling.

A 3D computational fluid dynamics model, similar to that presented by Xuet al. ((2004) Biorheology 41:113-125), was used to estimate theconcentrations of thrombus-derived macromolecules in blood that flowsover forming thrombus and transports the markers along the adjacentvessel wall distally. The model was based on the geometry shown in FIG.1A, with typical values for blood density and viscosity (Fung (1993)“Biomechanics: Mechanical Properties of Living Tissues” (ed 2nd). NewYork: Springer-Verlag). The model of local blood sampling wasimplemented using the finite element software ADINA (Watertown, Mass.).In addition, a 2D axisymmetric computational model, similar to that ofMarkou et al. ((1998) Annals Biomed. Engineering 26:502-511), was usedto estimate thrombosis marker distribution within the flow field.Computational modeling predicted that molecules of interest (βTG, fibrinD-dimer, and TAT) released or generated at sites of thrombus formation,would be concentrated (>99%) within a very thin peripheral bloodboundary layer (˜0.1 mm thick concentration boundary layer) along theimmediately distal vessel wall (data not shown). Thus, as employed here,the local sampling method effectively sampled the entire near-wallboundary layer region, immediately distal to the forming thrombus, forplatelet and coagulation markers of interest.

Flow Chamber Coagulation Studies.

Glass capillary tubes were coated with 100 μg/ml Horm collagen (NycomedArzenmittel, Munich, Germany). Whole human blood was collected into corntrypsin inhibitor (CTI, 40 μg/ml), discarding the first 1 ml to limitany activation of the coagulation pathway, and then perfused through thetubes at a shear rate of 265 s⁻¹ for 10 min. Prior to each experiment,blood was incubated with aXIMab (20 μg/ml), or PBS vehicle. In separateexperiments, washed platelets and RBCs were processed as described(McCarty et al. (2006) J. Thromb. Haemost. 4:1367-1378), and then mixedwith FXII deficient human plasma (<1% FXII, Haematologic Technologies,Essex Junction, Vt.) to reach a hematocrit of 40% and platelet count of300×10³/μl. The reconstituted blood was incubated with aXIMab (20 μg/ml)or CTI (40 μg/ml), recalcified with 7.5 mM CaCl₂ and 3.75 mM MgCl₂, andthen perfused immediately through collagen coated capillary tubes.Recalcified FXII deficient human plasma without RBCs or platelets wasalso perfused over collagen. Images were obtained by DIC microscopyafter three minutes of perfusion with modified Tyrodes buffer.

Data Analysis.

Mean values are given ±1 SEM (standard error of the mean). Occlusiondata were compared using the log-rank test. The two-tailed Student'st-test was used for all other single pair comparisons. A P value ≤0.05was considered significant.

Results

Inhibition of FXI by aXIMab In Vitro Prevents Fibrin FormationIndependent of FXIIa.

When whole human blood, which was anticoagulated with CTI to block thefunction of FXIIa, was perfused over collagen at arterial shear (265s⁻¹), platelets were deposited in large aggregates which becameenveloped by forming fibrin strands (FIG. 7B). In stark contrast, whenCTI blood was treated with aXIMab to inhibit FXI, fibrin deposition wasnoticeably attenuated with a modest reduction in platelet adhesion. Theoutcome was similar when using reconstituted FXII deficient blood, withor without CTI. Again, fibrin was generated independent of FXII, and FXIinhibition with aXIMab interrupted fibrin formation. No fibrin wasformed when recalcified FXII deficient human plasma was perfused overcollagen. For these experiments, human whole blood, anticoagulated withCTI (40 μg/ml) to inhibit FXIIa, or reconstituted FXII deficient humanblood was perfused through collagen coated capillary tubes at a shearrate of 265 s⁻¹ for 10 minutes. Prior to each experiment, blood wasincubated with either aXIMab (20 μg/ml), CTI (40 μg/ml) forreconstituted blood where indicated, or PBS vehicle. Images wereobtained via Kohler-illuminated Nomarski differential interferencecontrast (DIC) microscopy with a Zeiss Axiovert 200M microscope using aZeiss 63× oil immersion 1.40 NA plan-apochromat lens. Images werecaptured following three minutes of perfusion with modified Tyrodesbuffer using a Zeiss AxioCam with Slidebook 4.0 (Intelligent ImagingInnovations, Inc., Denver, Colo., USA). All experiments were performedat 37° C. Each image in FIG. 7B is representative of 2-3 experiments.

These data suggest that under physiologically relevant shear FXIpromotes fibrin formation independent of FXIIa in the presence ofcellular blood components.

Inhibition of FXI with aXIMab.

A baboon given aXIMab (1A6; 2 mg/kg) was followed for 4 weekspost-injection to assess FXI antigen (FXI:Ag), anticoagulant activity,and antibody inhibitor levels. FXI:Ag levels measured 1 h after aXIMabadministration had decreased from 110% to 40% of NBP levels, butsteadily increased thereafter, reaching 300% of control at day 8post-infusion (FIG. 8A). Plasma FXI antigen remained above 200% ofcontrol through day 15, after which there was a decrease to 130% by day27. FXI activity decreased from 125% to <1% 1 h after aXIMabadministration, and remained inhibited by >99% for 10 days afteradministration. The FXI activity slowly climbed thereafter, normalizingto 136% by day 27. Maximum circulating FXI inhibitor levels wereobserved at 1 h post aXIMab infusion (72 Bethesda units [BU]), whichdecreased to <0.6 BU by day 15 (FIG. 8B). Consistent with the FXI:AgELISA data, the 160 kDa band representing the FXI homodimer on SDS PAGEand Western blots of plasma increased in intensity between day 0 and 8(FIG. 8C). These results indicate that the anticoagulant effect ofaXIMab was due to its sustained presence in the circulation, and itsability to block FXI activation and/or activity, and not to clearance ofthe enzyme from the plasma. Since aXIMab separates from FXI during SDSgel electrophoresis, it remains unclear whether inhibition of FXI byaXIMab caused a rebound effect (increase in FXI secretion) or theFXI-aXIMab immune complexes were slowly cleared from the circulation.

Single i.v. bolus injections of aXIMab (2 mg/kg) were given to 4baboons. The FXI procoagulant activity was inhibited by 99.0%(98.8-99.5%) at 1 hr after infusion and remained inhibited >95% in allanimals for 8 days after administration (FIG. 2). No adverse reactionsto the antibody were observed. aPTT measurements were prolonged afteraXIMab administration to 65.6±2.0 sec compared with 30.5±0.7 sec incontrol animals, while the PT measurements were equivalent to PBSvehicle-treatment control values in the same animals (9.1±0.1 vs.9.0±0.1 sec, respectively, n=11 for each treatment). Plateletaggregation in platelet rich plasma in response to adenosine diphosphate(ADP) and collagen was unchanged for aXIMab-treated animals versuscontrol results.

aXIMab Treated Baboons are Protected from Collagen Initiated ThrombusDevelopment.

Previous studies have shown that FXI inhibition by a polyclonal antibodywas antithrombotic on tissue factor and contact initiating surfaces inprimates (Gruber & Hanson (2003) Blood. 102:953-955). Since vascularinjury can also expose large amounts of platelet reactive collagen toflowing blood, the effect of FXI inhibition on collagen initiatedthrombosis in baboons was examined. Significant differences in plateletaccumulation between aXIMab- and vehicle-treated groups were seen asearly as 10 min after graft exposure to blood flow (0.13±0.03×10⁹ versus0.23±0.03×10⁹ platelets, aXIMab versus control, P<0.05). The differencesremained statistically significant throughout the time course ofthrombus propagation (FIG. 3A). Graft platelet accumulation at 60 minwas 63% lower in aXIMab-treated animals than in vehicle controls(1.38±0.26×10⁹ versus 3.68±0.52×10⁹ platelets, aXIMab versus control,n=6 and 8, respectively, P<0.01; FIG. 3A). Systemic platelet countsprior to thrombosis studies were similar for both aXIMab and controlgroups (341±27×10³/μl versus 337±31×10³/μl, respectively), and did notchange significantly following thrombosis experiments. End-point fibrindeposition was reduced by 81% compared with controls (0.23±0.07 mg and1.18±0.19 mg, aXIMab versus control, P=0.001; FIG. 3B). Since aXIMab ismonospecific for the A3 domain of FXI, these data verify that FXI playsan important role in thrombus propagation under arterial-type flowconditions in primates.

Reduced Thrombin Generation and Platelet Activation During FXIInhibition.

Since inhibition of FXI could reduce thrombus formation in vivo both bylimiting thrombin-mediated platelet activation and fibrin formationand/or by increasing thrombolysis, levels of βTG, thrombin (TAT), andD-dimer were measured. Systemic pre-treatment βTG, TAT, and D-dimerlevels were comparable in vehicle- and aXIMab-treated baboons (19.3±2.6versus 24.8±3.8 IU/ml, 6.2±0.4 versus 4.6±0.6 μg/L, and 2.3±0.2 versus2.4±0.2 μg/ml, respectively). Surprisingly, we observed arobust >20-fold increase in TAT release into the blood stream from thegraft thrombus area, as measured in samples taken locally from thenear-wall region, immediately downstream of thrombus formation invehicle-treated baboons. Pre-treatment of baboons with aXIMab preventedthe increase in local TAT levels, indicating a considerable reduction inthrombin generation in the absence of FXI activity (FIG. 4A). TAT levelsin plasmas obtained by local sampling were reduced by up to 98% at 40min, while systemic TAT levels were reduced by 81% at 60 min versus theuntreated controls (4.3±0.5 versus 22.1±2.5 μg/L, n=6 and 7,respectively, P<0.001; FIG. 4B). Platelet activation at the thrombussurface, as assessed by the release of platelet α-granule βTG, wasreduced by 86% at 30 min in samples taken distal to thrombi in aXIMabtreated animals (FIG. 4C). Systemic βTG levels measured at 60 min werereduced by 42% by aXIMab (23.0±2.1 IU/ml, n=6) compared withvehicle-treated controls (39.5±5.5 IU/ml, n=7; FIG. 4D, P<0.05). LocalD-dimer levels were not changed at 60 min compared with baselinesystemic values in either control (2.3±0.2 and 2.4±0.4 μg/ml,respectively, n=7) or aXIMab treated animals (2.4±0.2 and 2.2±0.2 μg/ml,respectively n=6; FIG. 4E). Systemic D-dimer levels assessed at 60 minalso were unchanged from baseline systemic values in both groups(2.4±0.3 and 2.3±0.2 μg/ml, control versus aXIMab treated animals). Thisconfirms previous reports from similar baboon studies showing systemicD-dimer levels do not increase by 60 min after thrombus initiation,unless a thrombolytic agent is administered (Sundell et al. (1997)Circulation 96:941-948; Dichek et al. (1996) Circulation 93:301-309).

In order to assess the local sampling method for activation ofcoagulation and platelets, the system was tested without interposing acollagen coated thrombogenic device within the shunt loop. Although thetubing alone modestly increased the TAT and β-TG levels systemically andlocally (FIG. 4A-C), the elevations were not statistically significant.No fibrin formation or platelet deposition was measured on the controltubing.

These data indicate that: 1) FXI plays an important role in thrombingeneration and platelet activation during acute, arterial-type thrombusformation, and 2) endogenous local thrombolysis appears to be limitedduring the time course of these studies, regardless of FXI activity.

aXIMab Prevents Vascular Graft Occlusion.

Even though macroscopic thrombi formed rapidly in 4 mm i.d. vasculargrafts, none of these grafts occluded during 60 min of blood perfusion.The effects of inhibiting FXI activity on thrombus formation andocclusion were therefore evaluated using smaller diameter (2 mm i.d.),collagen-coated vascular grafts that accumulated thrombus under highershear conditions (wall shear rate=2120 s⁻¹). While the initial plateletaccumulation rate was similar in aXIMab-treated and vehicle-treatedbaboons, thrombus stability was profoundly reduced in the absence of FXIactivity. Within 12-15 min after initiation of blood perfusion thegrowth rate of platelet thrombus was reduced in all aXIMab-treatedanimals, and the number of platelets in the thrombus decreased abruptly,indicating reduced thrombus stability and net losses of thrombusmaterial to embolization (FIG. 5). Treatment with aXIMab prevented graftocclusion over 60 min in all experiments (5 of 5 grafts remainedpatent), compared with the results in vehicle-treated controls in which8 of 9 grafts occluded by 27.0±3.3 min (P<0.01; FIG. 5 and Table 1).Fibrin accumulation was also reduced by aXIMab treatment versus thevehicle-treated controls (0.18±0.02 versus 0.30±0.04 mg, respectively,P<0.01; Table 1). As expected, treatment with the positiveantithrombotic control, high dose ASA, reduced platelet deposition inthe 2 mm id. grafts, but did not completely interrupt occlusive thrombusformation. The time to graft occlusion was prolonged by aspirin by anaverage of 18 min in 4 grafts, while 2 additional grafts remained patentthroughout the 60 min study interval. High dose ASA was therefore lesseffective in preventing graft occlusion than aXIMab (P<0.05, aXIMabversus ASA). These data indicate that FXI plays an important role in thethrombogenic process that leads to acute occlusion of small calibervascular grafts.

TABLE 1 FXI inhibition by aXIMab prevents vascular graft occlusion inbaboons. Number Embolic events occluded per experiment Fibrin deposition(mg) Control 8/9 n.d. 0.30 ± 0.04 ASA 4/6 1.5 ± 0.6 0.23 ± 0.04 aXIMab0/5 6.6 ± 0.9  0.18 ± 0.02* Data shown are absolute values or means ±SEM when applicable. FXI, factor XI; aXIMab, anti-FXI monoclonalantibody; ASA, aspirin; n.d., none detected. *P < 0.01 compared withcontrol using the two-tailed Student's t-test.

No Antihemostatic Effects of FXI Inhibition with aXIMab.

Since all FDA approved anticoagulant and antiplatelet drugs produceunwanted antihemostatic effects, a new thrombosis specific therapy couldprovide a safe alternative for patients at a high risk of bleeding. Thetemplate bleeding is prolonged by both anti-platelet agents andanticoagulants in baboons (Gruber et al. (2007) Blood 109:3733-3740;Hanson et al. (1988) J. Clin. Invest. 81:149-158), yet FXI inhibition byaXIMab had no effect on the standard template bleeding time comparedwith vehicle-treated controls (3.5±0.3 versus 3.4±0.2 min, n=18 and 14,respectively). For comparison, single-dose ASA pre-treatment nearlydoubled the bleeding time to 6.4±0.7 min (n=10, P<0.01). No re-bleedingphenomena, petechiae, hematomas or other adverse bleeding events werenoted in any of the aXIMab- or ASA-treated animals during one weekfollow-up periods of observation. Taken together, these results showthat inhibition of FXI activity by a neutralizing monoclonal antibodymore effectively reduces acute occlusive arterial-type thrombuspropagation, with less effect on primary hemostasis, than anantihemostatic dose of aspirin in baboons.

Discussion

These studies demonstrate that FXI is crucial for the facilitatedproduction of robust amounts of thrombin on the flow surface of collageninitiated and propagating thrombi, which leads to platelet activation,fibrin formation, and thrombus stability under high and low arterialflow conditions. The antithrombotic/antiocclusive outcome of FXIinhibition was produced primarily through a substantial decrease inthrombin production rather than through an increase in fibrinolyticpathways. While not wishing to be bound by theory, since thrombin is themost potent physiological agonist of platelets, it is likely that thesignificant decrease in platelet activation observed during FXIinhibition resulted from the down regulation of thrombin generation.While FXI inhibition delayed platelet deposition onto collagen under lowarterial flow, early thrombus growth at high flow mirrored that ofcontrol studies. These results are consistent with the increasedefficiency of platelet GPIb binding to vWF on exposed collagen underhigh shear (Nishiya et al. (2002) Blood. 100:136-142), which producesless reliance on fibrin for early thrombus growth than under low shearconditions. In the propagation phase of thrombus growth however, thestability of thrombi exposed to high shear flow was robustly dependenton FXI and its ability to facilitate fibrin production and plateletactivation. Indeed, under high arterial flow, enhanced distalthromboembolization was distinctly noted, which completely preventedvascular graft occlusion in all aXIMab treated animals. These findingsare consistent with the antithrombotic and anti-occlusive phenotypeobserved in FXI deficient mice following ferric chloride-inducedarterial injury and thrombosis (Rosen et al. (2002) Thromb. Haemost.87:774-776; Wang et al. (2005) J. Thromb. Haemost. 3:695-702; Wang elal. J. Thromb. Haemost. 4:1982-1988). However, none of the previous invivo studies of flow-dependent thrombogenesis have provided evidence forthe underlying antithrombotic mechanism of FXI inhibition.

FXI promotes clot resistance to fibrinolysis through thrombin-mediatedactivation of the metaloproteinase TAFI, which proteolytically modifiesfibrin making it resistant to plasmin (Bajzar et al. (1996) J. Biol.Chem. 271:16,603-16,608; Broze & Higuchi (1996) Blood 88:3815-3823). Inaddition, inhibition of thrombin generation by anticoagulants enhancesclot lysis due to a slower forming, less dense fibrin network (Gruber etal. (1994) Blood 83:2541-2548; Nenci et al. (1992) Blood Coagul.Fibrinolysis 3:279-285). Both processes may account, at least in part,for the enhanced lysis of blood clots formed in the presence of ananticoagulant anti-factor XI antibody in the clamped jugular vein ofrabbits (Minnema et al. (1988) J. Clin. Invest. 101:10-14). During thepresent studies in baboons, the fibrinolytic degradation product D-dimerwas measured by local blood sampling in the vicinity of fibrin,platelet, and leukocyte rich arterial-type thrombi (Gruber et al. (2007)Blood 109:3733-3740). Unlike TAT and βTG levels, there were no changesdetected in local D-dimer for vehicle- or aXIMab-treated baboons. Thisfinding suggests that endogenous fibrinolysis may not be a dominantprocess on the surface of thrombi during acute arterial thrombogenesis,which is consistent with the observation that TAFI deficient mice thatwere expected to have a fibrinolytic phenotype were not protectedagainst injury-induced arterial thrombus formation (Nagashima et al.(2002) J. Clin. Invest. 109:101-110). Although the present study did notdemonstrate a role for fibrinolysis in rapid thrombus development, moreeffective endogenous fibrinolysis in the absence of FXI activity mayplay a role in later stages of thrombus development and reorganization.

The dramatic reduction in local TAT during FXI inhibition with aXIMabillustrates the importance of FXI in thrombin production on the surfaceof collagen initiated and propagating thrombi, which contributesdirectly to fibrin formation, platelet activation, and thrombusstability.

Since PPACK-thrombin cannot be detected in the enzyme immunoassaydescribed above, all local TAT was generated on the thrombus flowsurface or within the ˜1 cm distance from the thrombus to the PPACKinfusion port (minus the systemic contribution). A benefit of usingPPACK over other anticoagulants is its ability to rapidly inhibit anumber of active serine proteases of the coagulation pathway, includingfactor Xa, which, as a part of the prothrombinase complex on activatedplatelets can be shielded from other anticoagulants and continue togenerate thrombin. PPACK can also inhibit FXIa and limit unwantedcontact activation within the sampling syringe over the 10 min samplingintervals. Without PPACK infusion the local sampling port occludedwithin 20-30 min. Based on the CFD models employed, the majority of theinfused PPACK was captured within the sampling syringe. The small amountof PPACK that escaped into systemic circulation did not affect theplatelet or fibrin deposition onto the 4 mm collagen coated graftscompared with historical data. Also, the total infused PPACK, if givensystemically, is an order of magnitude less than that previously shownto prolong clotting times in baboons (Hanson et al. (1988) Proc. Natl.Acad. Sci. USA. 85:3184-188).

The decrease in local TAT and β-TG levels approaching 60 min in the notreatment collagen studies correlated with a decrease in plateletaccumulation rate. It is unknown what led to the pacification ofthrombus growth and mediator production, but is possibly the result ofactivated protein C (APC) generation or the production of othercoagulation inhibitors.

Another intriguing outcome from these experiments suggests thatcirculating tissue factor (TF), either through platelets ormicroparticles, does not appear to play a significant role in promotingthrombus development. Without wishing to be bound by theory, theformation of TF/FVIIa complex should support thrombin production andfibrin formation independent of the contact pathway, but inhibiting FXIsignificantly limits fibrin production on collagen. A likely scenario isthat FXIa promotes robust feedback amplification of thrombin, whichpromotes fibrin formation and platelet activation independent ofcirculating TF (Gailani et al. (1991) Science. 253:909-912; Broze et al.(1993) Thromb. Haemost. 70:72-74; Gailani et al. (2001) Blood.97:3117-3122; Yun et al. (2003) J. Biol. Chem. 48:48112-48119; Baglia etal. (2004) J. Biol. Chem. 279:49323-49329).

The activation of FXI by factor XIIa may also be an important processduring collagen initiated thrombus development.

The decrease in local TAT formation during FXI inhibition in baboons,combined with more limited platelet and fibrin accumulation, illustratesthe importance of continued FXI-dependent thrombin generation in thepropagation of thrombi. FXI dependent thrombin generation could occurvia continued FXI activation by FXIIa, thrombin, and/or autoactivation(Gailani & Broze (1991) Science 253:909-912; Broze & Gailani (1993)Thromb. Haemost. 70:72-74). Data appears to play an important role inexperimental thrombosis in mice (Renne et al. (2005) J. Exp. Med.202:271-281). However, the present in vitro flow chamber data presents amechanism whereby FXI promotes thrombosis independent of FXIIa duringshear flow conditions in the presence of blood cells. Althoughthrombin-mediated FXI activation in static plasma assays in vitro hasbeen questioned (Pedicord et al. (2007) PNAS USA 104:12,855-12,860), ourflow-augmented thrombosis model clearly demonstrates a FXII independentthrombogenic pathway for FXI.

Interestingly, while free FXI:Ag decreased to <1% for over a weekfollowing aXIMab injection, total FXI:Ag levels temporarily increasedseveral-fold above baseline following the disappearance of FXI activityin the baboon circulation. Whether this increase was due to a longerhalf-life of the circulating immune complexes or upregulation of FXIsynthesis and/or secretion remains to be explored.

The evolutionary role of FXI has been difficult to fully elucidate, butthese and other studies support a hypothesis that FXI functions, inpart, to promote hemostasis after transvacular injury, which can exposelarge amounts of collagen to flowing flood. In the absence of FXI,pro-hemostatic occlusive thrombi may fail to stabilize, leading toincreased and potentially fatal blood loss. Since tissue factordependent hemostatic processes dominate during most bleeding events, FXIlikely plays a role only after surgery or trauma, which are the dominantclinical manifestations of severe FXI deficiency in human patients.Moreover, since FXI seems to have its central effect on the flow surfaceof propagating thrombi, and patients with FXI deficiency have agenerally mild bleeding tendency, FXI appears to be a more thrombosisspecific treatment strategy than other existing antithrombotic methods,which can produce serious bleeding events independent of major trauma orsurgery.

Overall, the evidence presented above shows that FXI is critical forcollagen induced thrombus propagation by promoting a considerable localincrease in thrombin production, which in turn contributes to robustplatelet activation and fibrin formation. FXI inhibition however doesnot interfere substantially with primary hemostasis. Thus, therapeuticFXI inhibition represents an exceptionally promising treatment strategyto limit thrombus growth and thrombotic vessel occlusion (i.e. stroke,heart attack, DVT), while at the same time providing unmatchedhemostatic safety compared with other currently available antithromboticagents.

Example 2. Binding of the 1A6 Monoclonal Antibody to Human Factor XI

Results from a Western Blot study corroborating that anti-factor XImonoclonal antibody 1A6 recognizes Factor XI in human and non-humanprimate plasma is shown in FIG. 7A.

Comparative binding studies for monoclonal antibody 1A6 were conductedusing FXI, Prekallikrein (PK), and FXI/PK chimeras involving either thesubstitution FXI domains with PK domains (FIG. 9) or the substitution ofPK domains with FXI domains (FIG. 10). As shown in the right panel ofFIG. 9, monoclonal antibody 1A6 bound FXI but did not bind to PK. Inaddition, monoclonal antibody 1A6 bound all FXI/PK chimeras except thechimera in which the A3 domain of FXI was substituted with a PK domain(FIG. 9). For chimeras involving the substitution of PK domains withdifferent FXI domains, antibody 1A6 was shown to bind only to chimerasin which the A3 domain of FXI had been inserted (FIG. 10). From theseloss of function (FIG. 9) and gain of function (FIG. 10) studies, it wasdetermined that monoclonal antibody 1A6 bound to an epitope on the FXIA3 domain.

Binding studies were also conducted using recombinant FXI proteinsinvolving mutations of different amino acids within the A3 domain todetermine amino acids important for the binding of monoclonal antibody1A6. An example of the binding of monoclonal antibody 1A6 to a panel of25 such recombinant proteins in which 2 or 3 adjacent amino acids withinthe A3 domain were replaced with Alanine is shown in FIG. 11. Although apolyclonal anti-factor XI antibody bound to all FXI A3 domain mutants,monoclonal antibody 1A6 did not bind to some mutants (see FIG. 11).Based on such studies, amino acids important for the binding ofmonoclonal antibody 1A6 to the FXI A3 domain were determined, and areshown in FIG. 12 (amino acids 183 to 197, 203 to 204, 234 to 236, 241 to243, 252 to 254, and 258 to 260 of SEQ ID NO:1).

Example 3. Sequencing of the 1A6 Monoclonal Antibody and Coding Sequence

mRNA was extracted from a hybridoma cell line expressing the murineantibody known as Aximab (1A6), reverse transcribed, and antibodyspecific transcripts were PCR amplified. PCR products were cloned fordetermination of the nucleotide and amino acid sequences of the heavyand light chain variable regions of this antibody.

The cell line was successfully recovered from frozen stocks. mRNA wasextracted from the cell pellet of the hybridoma and RT/PCR was performedusing a system of degenerate primer pools. Heavy chain variable regionmRNA was amplified using a set of six degenerate primer pools (HA to HF)and light chain variable region mRNA was amplified using a set of eightdegenerate primer pools (LA to LH).

Aximab Heavy Chain: A strong DNA band of approximately the expected sizewas observed in primer pool HD. DNA from this band was purified andcloned, and eight clones were sequenced. Four of these clones aligned togive a functional, rearranged heavy chain (Table 2, FIG. 13A).

TABLE 2 Aximab antibody (1A6) sequence analysis (CDR definitions andsequence numbering according to Kabat). H Chain L Chain CDR 1 Length 7aa 15aa  CDR 2 Length 16aa 7aa CDR 3 Length 14aa 9aa Closest HumanGermline* IGHV2-5 (76%) IGKV4-1 (70%) Closest Human FW1* IGHV2-5 (80%)IGKV4-1 (83%) Closest Human FW2* IGHV2-5 (86%) IGKV4-1 (100%) ClosestHuman FW3* IGHV2-70 (72%) IGKV3-11 (78%) Closest Human J* IGHJ6 (92%) J4(92%) Max No. Mouse 9 (2) 5 (3) FR Residues** *Germline ID(s) indicatedfollowed by % homology. **Indicates maximum number of mouse residuesthat need to be sourced from human sequence segments with number ofthose potentially critical for affinity indicated in brackets.

Aximab Light Chain:

Strong DNA bands of the expected size were observed in primer pools LB,LC and LG. DNA from each band was purified and cloned, and four clonesfrom each band were sequenced. The eight clones from pools LB and LCwere found to align with the well described aberrant kappa transcriptfound in some hybridomas. The four clones from pool LG aligned to give afunctional, rearranged light chain (Table 2, FIG. 13B).

The heavy and light chain variable region sequences from the Aximabhybridoma were determined. It was confirmed that Aximab is a murineIgG/kappa antibody and that both chains of this antibody have closesequence homologues in the human antibody databases through theframework regions (Table 2). This should facilitate subsequenthumanization by either Composite Human Antibody™ technology orCDR-grafting. Heavy and light chain clones of each antibody wereproduced and are therefore available for humanization. ExemplaryCDR-grafted heavy and light chain variable region sequences are shown inFIGS. 13C and 13D.

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

The invention claimed is:
 1. A method for inhibiting thrombosis withoutcompromising hemostasis in a subject in need thereof, comprisingadministering to the subject a therapeutically effective dose of ananti-Factor XI (FXI) antibody or biologically active fragment thereof,wherein the antibody or biologically active fragment thereof comprises:a variable heavy (V_(H)) domain comprising three complementaritydetermining regions (CDRs), wherein the three CDRs comprise the threeCDR sequences of SEQ ID NO: 3 or SEQ ID NO: 7; and a variable light(V_(L)) domain comprising three CDRs, wherein the three CDRs comprisethe three CDR sequences of SEQ ID NO: 5 or SEQ ID NO:
 9. 2. The methodof claim 1, wherein at least one of the three CDRs of the V_(H) domaincomprises the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 11, orSEQ ID NO:
 12. 3. The method of claim 2, wherein the three CDRs of theV_(H) domain comprise the amino acid sequences of SEQ ID NO: 10, SEQ IDNO: 11, and SEQ ID NO:
 12. 4. The method of claim 1, wherein at leastone of the three CDRs of the V_(L) domain comprises the amino acidsequence of SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO:
 15. 5. Themethod of claim 4, wherein the three CDRs of the V_(L) domain comprisethe amino acid sequences of SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO:15.
 6. The method of claim 1, wherein the amino acid sequence of theV_(H) domain is at least 90% identical to SEQ ID NO: 3 or SEQ ID NO: 7.7. The method of claim 1, wherein the amino acid sequence of the V_(L)domain is at least 90% identical to SEQ ID NO: 5 or SEQ ID NO:
 9. 8. Themethod of claim 1, wherein the amino acid sequence of the V_(H) domaincomprises SEQ ID NO: 3 or SEQ ID NO:
 7. 9. The method of claim 1,wherein the amino acid sequence of the V_(L) domain comprises SEQ ID NO:5 or SEQ ID NO:
 9. 10. The method of claim 1, wherein the thrombosis isassociated with myocardial infarction, unstable angina, atrialfibrillation, stroke, renal damage, pulmonary embolism, deep veinthrombosis, percutaneous translumenal coronary angioplasty, disseminatedintravascular coagulation, sepsis, artificial organs, shunts, orprostheses.
 11. The method of claim 1, further comprising administeringthe antibody in combination with an antithrombotic agent.
 12. The methodof claim 11, wherein the antithrombotic agent is a direct or indirectthrombin inhibitor, a Factor X inhibitor, a Factor IX inhibitor, aFactor XII inhibitor, a Factor V inhibitor, a Factor VIII inhibitor, aFactor XIII inhibitor, a Factor VII inhibitor, a tissue factorinhibitor, a profibrinolytic agent, a fibrinolytic or fibrinogenolyticagent, a carboxypeptidase B inhibitor, a platelet inhibitor, a selectiveplatelet count reducing agent, or a Factor XI inhibitor.
 13. The methodof claim 1, wherein the subject is human.
 14. The method of claim 1,wherein the antibody or biologically active fragment thereof isadministered to the subject in a single dose of about 0.01 mg/kg toabout 10 mg/kg.
 15. The method of claim 14, wherein the antibody orbiologically active fragment thereof is administered to the subject in asingle dose of about 2 mg/kg.
 16. The method of claim 1, wherein theantibody or biologically active fragment thereof is administered to thesubject via systemic, topical, parenteral, or enteral administration.17. A method for inhibiting thrombosis without compromising hemostasisin a subject in need thereof, comprising administering to the subject atherapeutically effective dose of a pharmaceutical composition, thepharmaceutical composition comprising a pharmaceutically acceptablecarrier and an anti-Factor XI (FXI) antibody or biologically activefragment thereof, wherein the antibody or biologically active fragmentthereof comprises: a variable heavy (V_(H)) domain comprising threecomplementarity determining regions (CDRs), wherein the three CDRscomprise the three CDR sequences of SEQ ID NO: 3 or SEQ ID NO: 7; and avariable light (V_(L)) domain comprising three CDRs, wherein the threeCDRs comprise the three CDR sequences of SEQ ID NO: 5 or SEQ ID NO: 9.18. The method of claim 17, wherein the pharmaceutical composition isformulated for immediate release of the antibody or biologically activefragment thereof.
 19. The method of claim 17, wherein the pharmaceuticalcomposition is formulated for controlled release of the antibody orbiologically active fragment thereof.